Parastrigea diovadena Dubois and Macko, 1972, is an allogenic trematode species that infects the intestine of white ibis. This widely distributed Neotropical species has been studied poorly, and nothing is known about its population genetic structure. In the current study, we attempt to fill this gap for the first time and to explore the genetic diversity in P. diovadena populations from three biogeographic provinces (Sierra Madre Oriental, Sierra Madre Occidental, and Sierra Madre del Sur) in the Neotropical region of Mexico. Newly generated sequences of the internal transcribed spacers (ITS) from ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA were compared with sequences available from the GenBank data set. Phylogenetic analyses performed with the ITS and cox 1 data sets using maximum likelihood and Bayesian inference unequivocally showed that new sequences of P. diovadena recovered from the white ibis formed a clade with other sequences of specimens previously identified as P. diovadena. The intraspecific genetic divergence among the isolates was very low, ranging from 0 to 0.38% for ITS and from 0 to 1.5% for cox 1, and in combination with the phylogenetic trees confirmed that the isolates belonged to the same species. The cox 1 haplotype network (star-shaped) inferred with 62 sequences revealed 36 haplotypes. The most frequent haplotype (H3, n = 18) corresponded to specimens from all the populations (except Tecolutla, Veracruz). In addition to the common haplotype, we identified four other shared haplotypes (H2, H9, H12, and H14) and 31 unique haplotypes (singlets). In addition, high haplotype diversity (Hd = 0.913), low nucleotide diversity (Pi = 0.0057), and null genetic differentiation or population structure (Fst = 0.0167) were found among the populations from the three biogeographic provinces. The results suggest that the biology of the definitive host has played a key role in the population genetic structure of Parastrigea diovadena in the Neotropical region of Mexico.