The fish genus
Poeciliopsis
constitutes a valuable research system for evolutionary ecology, whose phylogenetic relationships have not been fully elucidated. We conducted a multilocus phylogenetic study of the genus based on seven nuclear and two mitochondrial loci with a thorough set of analytical approaches, that is, concatenated (also known as super‐matrix), species trees, and phylogenetic networks. Although several relationships remain unresolved, the overall results uncovered phylogenetic affinities among several members of this genus
.
A population previously considered of undetermined taxonomic status could be unequivocally assigned to
P. scarlli
; revealing a relatively recent dispersal event across the Trans‐Mexican Volcanic Belt (TMVB) or Pacific Ocean, which constitute a strong barrier to north–south dispersal of many terrestrial and freshwater taxa. The closest relatives of
P. balsas
, a species distributed south of the TMVB, are distributed in the north; representing an additional north–south split in the genus. An undescribed species of
Poeciliopsis
, with a highly restricted distribution (i.e., a short stretch of the Rio Concepcion; just south of the US‐Mexico border), falls within the
Leptorhaphis
species complex. Our results are inconsistent with the hypothesis that this species originated by “breakdown” of an asexual hybrid lineage. On the other hand, network analyses suggest one or more possible cases of reticulation within the genus that require further evaluation with genome‐wide marker representation and additional analytical tools. The most strongly supported case of reticulation occurred within the subgenus
Aulophallus
(restricted to Central America), and implies a hybrid origin for
P. retropinna
(i.e., between
P. paucimaculata
and
P. elongata
). We consider that
P. balsas
and
P.
new species are of conservation concern.