The accurate identification of insect has a primordial role for allocation to specific pests that affecting crops, being the morphological similarities of many species a great challenge faced by entomologist, is why DNA-based methodologies are used as a supplemental means of morphological method. Asparagus (Asparagus officinalis) is the most important crop in Peru, in which, Prodiplosis sp. (Diptera: Cecidomyiidae) polyphagous insect is a major pest which reduces its production to 50%. In the present study the mtDNA of Prodiplosis sp. used for the molecular identification and diversity analysis into 34 larvae colected from Asparagus officinalis, Capsicum annuum and Citrus spp. crops. 490 bp and 712 bp fragments of cytochrome oxidase I gene were amplified using polymerase chain reaction followed by direct sequencing. Nucleotide sequence divergences were calculated using the distance Kimura two parameters model showed the formations of two separates clades grouped either host. Genetic diversity in the total population, within populations and among populations was of 0.019, 0.005 and 0.014, respectively showed lowest divergence, variability within groups and among groups was 26.7% and 73.3%. The AMOVA analysis within populations (-1.13%) and statistic value FST (0.01129) was low, which is explained by high gene flow Nm (43.79), suggesting that crops are affected by two haplotypes (h1: TC y h2: GT) of the same pest. In conclusion, based to a PCR sensibility and specificity and mtDNA sequence information, it is suggest that the proof would be a molecular tool DNA-based complementary to the morphological identification that can be applied in studies of genetic diversity of Prodiplosis sp. in other crops.