Correct species identification is the first and the most important criteria in entomological evidence-based postmortem interval (PMI) estimation. Although morphological keys are available for species identification of adult blow flies, keys for immature stages are either lacking or are incomplete. In this study, cytochrome oxidase subunit 1 (COI) reference data were developed from nine species (belonging to three subfamilies, namely, Calliphorinae, Luciliinae, and Chrysomyinae) of blow flies from India. Seven of the nine species included in this study were found suitable for DNA-based identification using COI gene, because they showed nonoverlapping intra- (0.0–0.3%) and inter-(1.96–18.14%) specific diversity, and formed well-supported monophyletic clade in phylogenetic analysis. The remaining two species (i.e., Chrysomya megacephala (Fabricius) and Chrysomya chani Kurahashi) cannot be distinguished reliably using our database because they had a very low interspecific diversity (0.11%), and Ch. megacephala was paraphyletic with respect to Ch. chani in the phylogenetic analysis. We conclude that the COI gene is a useful marker for DNA-based identification of blow flies from India.