Magister Artium - MA / Introduction A high burden of Human Immunodeficiency Virus (HIV) constitutes a key global public health concern. In South Africa, it is estimated that 260 000 children aged 0-14 years had HIV infection and only 63% of them were reported to have received HIV treatment in 2018. Without antiretroviral therapy (ART), HIV infection during infancy is associated with rapid disease progression where more than half of all infected children are expected to die before two years of age. Early infant diagnosis (EID) of HIV is therefore essential for accessing timely HIV treatment. However, preanalytical errors within the EID diagnostic cascade prevent optimal access to HIV polymerase chain reaction (PCR) results. The aim of this study was to describe the prevalence and contributing factors of preanalytical errors resulting in missed diagnostic opportunities for HIV among children below 18 months of age in Thabo Mofutsanyana (TM) district. Methodology The study was conducted using a descriptive cross-sectional study design and data was collected in two phases. Phase 1 involved obtaining the routine HIV PCR testing data set from the National Health Laboratory Services (NHLS) for all samples collected at TM public health facilities in 2018 and registered by NHLS. Phase 2 included a facility assessment checklist and semi structured questionnaire administered to 36 health care workers (HCWs) from 10 purposively selected health facilities. Data collected in phase 2 was analyzed to describe health facilities and HCW factors that might be contributing to the HIV PCR preanalytical errors. Results Phase 1. Of the 9318 samples included in the analysis, 49.6% were birth HIV PCRs whilst 42.1% and 8.3% were from 10 weeks and above 12 weeks age categories, respectively. A total of 745 (8%) samples were rejected because of the following preanalytical errors: insufficient specimen (84.3%), unsuitable sample (9.9%) and clerical error (5.8%).By age, the preanalytical errors were: birth (534), 10 weeks (170) and the above 12 weeks age category (41). Hospitals had the highest proportion of total preanalytical errors (58.1%). For PHCs the errors were: insufficient specimen (90%), unsuitable sample (5.5%) and clerical (4.8%).