Health related problems due to vitamin A deficiency affect a large part of the world’s population. Enhancing the levels of vitamin A in staple foods is regarded as a sustainable food-based approach that can have a significant long term beneficial impact on optimising vitamin A intake. Banana and plantain (Musa spp.) is the staple food crop for millions of people in Africa and other parts of the world. Increasing the nutritional qualities of Musa spp. will translate into improved diets. This study used high-performance liquid chromatography (HPLC) to determine the β-carotene content of 47 banana genotypes from the International Institute of Tropical Agriculture (IITA) germplam collection in Uganda and used a color meter to assess the correlation between pulp color intensity and β-carotene levels. There was wide variability in β-carotene levels within and among the different groups of banana studied. Banana genotypes from Papua New Guinea (PNG) had the highest levels of β-carotene with values as high as 2594.0 μg/100 g edible pulp. A positive correlation existed between pulp color intensity and β-carotene concentration. Accessions with relatively high levels of β-carotene,especially the PNG genotypes, could be deployed to regions with high vitamin A deficiency and/or be used as parents for development of vitamin dense varieties. The PNG genotypes could be useful in genetic studies related to vitamin A in banana.