The colonization of decomposing remains by necrophagous insects such as blow flies is of forensic importance because the progression through the various stages of insect development can be correlated to time of death. The ability to infer this information hinges on accurate determination of the fly species that are associated with the entomological evidence collected. This evidence can include eggs, larvae, pupae, and puparial casings. Determination of the egg’s identity is particularly challenging because the eggs of multiple species are morphologically very similar. We report here that the species identity of fly eggs can be determined from their chemical fingerprint signatures acquired by direct analysis in real time high-resolution mass spectrometry (DART-HRMS). Thus, freshly laid eggs were collected and readily distinguished from multiple necrophagous fly species in the Manhattan area of New York City. These species included representatives from the blow fly family (Calliphoridae), specifically Calliphora vicina, Lucilia sericata, L. coeruleiviridis, and Phormia regina species as well as the Phoridae and Sarcophagidae families. Multivariate statistical analysis of their observed DART-HRMS spectra revealed intraspecies similarities and interspecies differences that were the basis of species differentiation. The chemical basis of discrimination was differences in amino acid profiles. This represents the first chemically based fly egg identification approach with applications to forensic entomology. The rapidity of the method makes feasible the generation of a fly egg chemical profile database against which the DART-HRMS spectra of unknown eggs can be screened to rapidly assess species identity without needing to rear the eggs to adulthood.