Kangaroo mice (Microdipodops) inhabiting the Mono Basin and adjoining valley regions of California and Nevada represent a disjunct distributional isolate and have been considered as a distinct species or, more recently, as 2 subspecies of M. megacephalus. Analysis of patterns of geographic variation in 11 populations in the Mono Basin region shows that kangaroo mice inhabiting the northern portion of this peripheral isolate are relatively large and dark (referred to as M. m. nasutus), and those to the southern end are small and pale (termed M. m. polionotus), and a cline exists between the 2. Inasmuch as several morphological characters are correlated positively with environmental measures (e.g., hind-foot length and climatic severity; pelage color and soil color), it appears that variation in morphology is responding to a selection gradient. Mitochondrial DNA sequence data, together with chromosomal and protein information, reveal that the 2 subspecies are nearly identical genetically. Moreover, molecular phylogeographic analysis reveals that the Mono kangaroo mice belong to the southeastern geographic unit of M. megacephalus (a mean of 1.86% sequence divergence) and are genetically most close to animals from the San Antonio locality (more than 100 km to the east). It is hypothesized that distributional shifts in the geographic range of kangaroo mice in response to climatic fluctuation during the late-Pleistocene and Holocene times resulted in the westward expansion and eventual colonization and isolation of kangaroo mice in the Mono Basin region. Our vicariant biogeographical interpretation suggests a historical route through the Lahontan Trough and the physiographic discontinuity east of Mono Lake (i.e., between the Wassuk Range and the White Mountains) that may serve as a biogeographic model for other basin-dwelling organisms. Lastly, the systematic status of the 2 subspecies is evaluated; a single subspecies, polionotus, is recognized, with nasutus placed in synonymy.