Biofortification is a promising, cost effective and sustainable strategy to mitigate micronutrient deficiencies and related health complications. The principal aims of the study encompassed the quantification of the carotenoid content of 147 maize inbred lines, the identification of variable regions within the ZEP1 gene, the correlation of these observed variances in the presence of this gene with carotenoid content, and to identify lines harboring the favorable alleles for the crtRB1 gene. The correlations observed among the carotenoids synthesized by specific branch of the biosynthetic pathway were both significant and positive. Utilizing gel-based genotyping, 24 lines with contrasting carotenoid profiles were selected, evaluated and sequenced. Analysis of the variation in the sequence classified these lines into distinct groups. The findings highlight that inbred lines both group 1 and group 8 exhibited significant associations with the carotenoid content of the lines. Specifically, ZEP1_7852, a discernible variation belonging to group 8, was significantly associated with zeaxanthin content and total carotenoid content. Twenty-five inbred lines with provitamin A content surpassing 15 μg/g that harbor the favorable alleles of the crtRB1 gene were identified using KASP SNP zm0016. These lines can be used as donor parents for further enhancement of provitamin A in maize.