Background: Biofortification of staple crops is a cost effective and sustainable approach that can help combat vitamin A and other micronutrient deficiencies in developing countries. PCR -based DNA markers distinguishing alleles of three key genes of maize endosperm carotenoid biosynthesis (PSY1, lcyE and crtRB1) have been developed to facilitate maize provitamin A biofortification via marker assisted selection. Previous studies of these functional DNA markers revealed inconsistent effects. The germplasm previously employed for discovering and validating these functional markers was mainly of temperate origin containing low frequencies of the favourable allele of the most significant polymorphism, crtRB1-5'TE. Here, we investigate the vitamin A biofortification potential of these DNA markers in a germplasm panel of diverse tropical yellow maize inbred lines, with mixed genetic backgrounds of temperate and tropical germplasm to identify the most effective diagnostic markers for vitamin A biofortification.Results: The functional DNA markers crtRB1-5'TE and crtRB1-3'TE were consistently and strongly associated with provitamin A content across the tropical maize inbred lines tested. The alleles detected by these two functional markers were in high linkage disequilibrium (R-2 = 0.75) and occurred in relatively high frequency (18%). Genotypes combining the favourable alleles at the two loci (N = 20) displayed a 3.22 fold average increase in beta-carotene content compared to those genotypes lacking the favourable alleles (N = 106). The PSY1 markers were monomorphic across all of the inbred lines. The functional DNA markers for lcyE were associated with lutein, and with the ratio of carotenoids in the alpha and beta branches, but not with provitamin A levels. However, the combined effects of the two genes were stronger than their individual effects on all carotenoids.Conclusions: Tropical maize inbred lines harbouring the favourable alleles of the crtRB1-5'TE and 3'TE functional markers produce higher levels of provitamin A. Such maize lines can be used as donor parents to speed up the development of provitamin A biofortified tropical maize varieties adapted to growing conditions and consumer preferences, providing a route towards mitigation of vitamin A malnutrition in Sub-Saharan Africa.