In our laboratory, DNA sequencing by Sanger method is used as the “gold standard” for clinical diagnostics, microbe identification (bacteria and yeast, mainly) and genome characterization. In this research, we used it to characterize a conflicting locus in a Saccharomyces cerevisiae sequencing project. When sequenced, the resulting electropherogram of the analyzed locus showed a pattern indicating either sample contamination or allele variation. Molecular cloning was chosen as the most straight-forward strategy to solve the dilemma. The initial characterization of recombinant plasmids by restriction enzyme digestion confirmed the presence of two genomic sequences. Their Sanger sequencing revealed two alleles distinguishable by a total of 29 nucleotide differences (25 of which were SNPs). NCBI BLAST revealed that the conflicting locus covered an intergenic region and a coding sequence for a putative permease protein. The present study shows the utility of the classical molecular cloning technique to solve problems of modern genome projects.