Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612 bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343 bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170 bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810 bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.