Provitamin A (Pro-VA) is necessary for human vision and immune system health, especially in growing children. The first committed step in the maize carotenoid biosynthesis pathway is catalyzed by phytoene synthase 1 (encoded by PSY1) which controls the flux of substrates into the pathway. The flow of these substrates could be directed into production of the β-branch carotenoids (the step controlled largely by the lycopene epsilon cyclase gene), but terminated after the production of β-carotene, rather than allowing it to be converted into the next metabolite (the step controlled largely by the β-carotenoid hydroxylase gene). In this study, PSY1 was subjected to association mapping in two diverse maize populations, quantitative trait loci (QTL) mapping in one segregating population, and expression analysis of lines polymorphic for sites within PSY1. The results indicated that a 378-bp InDel upstream of the transcription start site and a SNP in the fifth exon resulting in a Thr to Asn substitution, explaining 7 and 8 % of the total carotenoid variation, respectively, may be functional sites associated with total carotenoid levels in maize grain. Analysis of the evolution of PSY1 strongly suggests that there was positive selection for these polymorphic sites after the divergence of yellow maize from white maize.