This study is part of an NRF sponsored research project entitled “Neotectonics and its applications for the exploration of groundwater in the fractured Karoo aquifers in the Eastern Cape” under the NRF Niche area of Water Resources Management and Sustainable Development in the Eastern Cape Province. The identification of relatively highly productive wells in the Karoo fractured aquifers is extremely difficult. This study aims to identify neotectonic zones and lower stress fields, and apply the results to groundwater exploration in the Eastern Cape Province. The methodologies adopted in this study include: a comprehensive literature review, extensive field mapping and investigation such as road cuts, sampling for laboratory studies, examination of seismic data, study of hot and ordinary springs, and interpretation of aerial photography and satellite images. Three main neotectonic belts were identified in the Eastern Cape (southern neotectonic belt, northern neotectonic belt and eastern neotectonic belt) based on literature review and field interpretations. The southern neotectonic belt (from the Cape Fold Belt to the lower Beaufort Group boundary) is characterized by the reactivation of the Coega-Bavianskloof and Sauer faults, the presence of a hot spring near Fort Beaufort, the slickenlines and discrete slickenlines and specifically the seismic events that were recorded in the Eastern Cape from 1850 to 2007. In this southern neotectonic belt the remote sensing has also revealed the presence of the Fort Beaufort fracture, the quartz veins seen in some dolerites and the different vegetation types along it may indicate that this fracture is possibly a fault; moreover the Quaternary sediments and weathered dolerites indicate that the Fort iii Beaufort fracture is characterized by groundwater circulation and accordingly is a good target for groundwater exploration, this fracture is a post-Karoo structure and possibly a neotectonic feature. In addition, the kaolin deposit, chiefly developed in the Dwyka tillite near Grahamstown is clearly controlled by neotectonic fracture zones. The northern neotectonic belt near the country of Lesotho is marked by the presence of the Senqu seismotectonic regime and hot springs. The Quaternary Amatole-Swaziland (formerly Ciskei-Swaziland) axis of uplift makes the eastern part of the province the third neotectonic zone, the asymmetric meanders of the Mbashe river in the vicinity of Qunu near Mthatha derived possibly from this Quaternary uplift; this asymmetric feature of meanders implies that the river has tried to maintain stability of its valley where tilting occurred. Within these neotectonic belts the central part of the Eastern Cape may be considered a static and inactive belt. A northwesterly trend for the maximum principal compresssional stress predominates in the Eastern Cape and is correlated with the present major structural control of the province. The current stress regime determination was derived from faults, joints and quartz veins only on kaolin deposits. Systematic joints reflect regional tectonic stress trajectories at the time of fracturing. Discharge rates of groundwater from boreholes as provided by the Department of Water and Forestry were used to confirm and predict water exploration targets. The region of Tabankulu (near Kwazulu Natal) in the northern neotectonic belt has remarkable discharge rates of groundwater (11.1 l/s, 4.65 l/s, 6.49 l/s, 42 l/s). The region of Mthatha, nearly surrounding the Amatole-Swaziland axis (former Ciskei-Swaziland iv axis) of uplift which might have generated some new faults, has a number of springs. These two regions should serve as case studies for future research. Apart from these two regions, two others regions can be considered as case studies for future groundwater exploration targets: the Bath Farm hot spring near the Fort Beaufort neotectonic fault and the vicinity of what is known as the Fort Beaufort fracture near Teba and Cimezile villages 20km north west of Fort Beaufort. It is concluded that the study of neotectonics and stress fields may be a useful tool for groundwater exploration in the Karoo fractured aquifers in the Eastern Cape, and in similar regions elsewhere in South Africa and in Africa.