We analyzed morphometric and molecular variation among 8 populations of Peromyscus zarhynchus grouped into 5 pooled samples representing separate physiographic regions across the range of this species in Chiapas, Mexico, and western Guatemala. Mitochondrial sequence data identify 2 well-supported and reciprocally monophyletic clades, separating all Chiapas specimens from those in Guatemala. These 2 clades group as a strongly supported monophyletic lineage aligned with other members of the Peromyscus mexicanus species group. The Chiapas clade is further subdivided into 4 subclades: 1) samples from the western part of the state, 2) specimens from a single locality in Northern Chiapas, 3) all central localities, and 4) those from a single locality in Eastern Chiapas. The molecular distance in the mitochondrial cytochrome-b gene (Cytb) between the 2 major clades is relatively low (mean p-distance = 3.66%); those between the 4 Chiapas subclades are even less (mean p-distance 2.73%). Multivariate analyses of external and craniodental morphometric variables also distinguish 2 major groups, separating Guatemalan from Chiapas samples but with the latter also divided into 2 subgroups, one that segregates the Northern Chiapas sample from those distributed elsewhere in that state. The Guatemalan and Chiapas samples differ in both cranial size and shape variables. The second-level separation of samples from within Chiapas (northern versus all others) is interpreted to result from the combination of local adaptation to distinct physiographic regions and geographic isolation generated by patches of suitable habitat. We describe the Guatemalan samples as a distinct species based on their molecular and morphological uniqueness, and argue that P. zarhynchus itself is divided into definable subspecies, with the nominotypical form P. z. zarhynchus, restricted to the vicinity of its type locality (Tumbalá) in Northern Chiapas, and P. z. cristobalensis with type locality of San Cristobal, over the remainder of the species range in the state.