Sweet corn is a popular food worldwide. Traditional shrunken2 (sh2)-derived sweet corn possesses low lysine (0.150–0.250%), tryptophan (0.030–0.040%) and provitamin-A (proA: 3–4 μg/g). Here transcript profiles of a newly developed 12 sh2-based sweet corn inbreds possessing mutant opaque2 (o2) and β-carotene hydroxylase (crtRB1) genes were analyzed during three stages of kernel development viz., 20-, 24- and 28- days after pollination (DAP). Introgressed inbreds had higher lysine (0.309%), tryptophan (0.077%) and proA (17.28 μg/g). Across stages of kernel development, transcript levels of mutant sh2, o2 and crtRB1 genes were significantly lower than the wild type (sh2+, o2+ and crtRB1+) alleles. Among genes, sh2 had the highest level of transcripts accumulation followed by crtRB1 and o2. While o2 and crtRB1 transcripts attained peaks at 24-DAP, the highest peak for sh2 was recorded at 28-DAP. Strong negative correlation (r = −0.99 to −0.71) was observed between the transcripts and corresponding nutrients. The study provided insight of complex regulation of sh2, o2 and crtRB1 genes during various stages of kernel development. This is a first of its kind study to analyze the transcript level variation of mutant allele of sh2, o2 and crtRB1 genes among novel biofortified sweet corn genotypes.