Potato cultivars exhibit a wide variation in skin and flesh colour due to the presence of pigments. This study established that potato cultivars differ greatly with respect to types and concentrations of carotenoids in tubers. A total of 46 cultivars were evaluated for quantitative and qualitative carotenoid composition in different growing seasons, locations, storage conditions and disease symptoms. Factors controlling carotenoid accumulation were also tested by developing an in vitro minituber system as a new high-throughput model system for carotenogenesis in potato tubers. Tuber flesh colour was found to correlate with total carotenoid content in potato cultivars grown in both New Zealand and Netherlands. The main carotenoids identified in 32 potato cultivars in New Zealand were lutein, neoxanthin, violaxanthin and β-carotene. The ratio of these carotenoids varies between cultivars. Neoxanthin was detected in only 13 cultivars (10.59 to 69.21µg/g DW); violaxanthin was found only in 1 cultivar (32.76 µg/g DW). Whereas lutein and β-carotene were found in most of the cultivars but the concentration varied from (0.00 to 160.63 µg/g DW) and (0.00 to 13.62 µg/g DW) respectively. The main carotenoids identified in 12 cultivars grown in the Netherlands were neoxanthin, violaxanthin and lutein, whereas zeaxanthin was not found in any of the cultivars analysed. Marked differences were observed between the same potato cultivars grown in New Zealand and the Netherlands. Therefore cultivars were analysed over a second growing season to assess stability in carotenoids composition. The carotenoid profiles of the potato tubers grown for two different seasons showed highly significant differences between the cultivars, the seasons, the carotenoid pigments, and all combinations of interactions, indicating the complex nature of factors influencing carotenoid composition. Reflectance colorimeter measurement of yellow hue component in this study confirmed that the higher the total carotenoid content, the greater the yellow intensity colour. Eight cultivars were grown at three locations in New Zealand and Agria and Desiree were grown at eight locations in the Netherlands to further investigate the stability of carotenoid composition. Highly significant differences were observed between the cultivars, the locations, the carotenoid pigments, and all combinations of interactions, which emphasises that changes in carotenoid composition are complex and the responses are not consistent across cultivars. Reflectance colorimeter measurement of yellow hue component confirmed the relationship between the yellow colour intensity of tuber flesh, as well as confirming the interaction between colour and locations. Disease and post harvest storage conditions markedly influenced the levels of total carotenoid, neoxanthin, violaxanthin, zeaxanthin, lutein and β-carotene in potatoes. The magnitude of these effects depends on the cultivar, time of storage, and the intensity of powdery scab symptoms. Results showed that long term storage resulted in the accumulation of neoxanthin, violaxanthin and zeaxanthin with a concomitant decreased of lutein, β-carotene and total carotenoid content. Genotypes infected with disease (lower and higher scab score) resulted in accumulation of violaxanthin, β-carotene and total carotenoid with a concomitant decreased in neoxanthin and lutein. A high-throughput model system for investigating carotenoid biogenesis in potato tubers was developed. This involved in vitro potato minitubers and was validated by assessing the effects of environmental variables, such as drought stress, light intensity and nutrient availability on carotenoid accumulation. Light influenced the presence of zeaxanthin, whereas water stress and nutrient strength influenced the accumulation of neoxanthin and violaxanthin. Although these factors had an effect on the carotenoid content and profile, the most influential factor appeared to be cultivar selection.