Ponderosa pine (Pinus ponderosa) is among the most broadly distributed conifer species of western North America, where it possesses considerable ecological, esthetic, and commercial value. It exhibits complicated patterns of morphological and genetic variation, suggesting that it may be in the process of differentiating into distinct regional lineages. A robust analysis of genetic variation across the ponderosa pine complex is necessary to ensure the effectiveness of management and conservation efforts given the species’ large distribution, the existence of many isolated disjunct populations, and the potential susceptibility of some populations to climate change and other threats. We used highly polymorphic nuclear microsatellite markers and isozyme markers from 3113 trees in 104 populations to assess genetic variation and structure across the geographic range of ponderosa pine. The results reveal pervasive inbreeding and patterns of genetic diversity consistent with the hypothesis that ponderosa existed in small, as-yet-undetected Pleistocene glacial refugia north of southern Arizona and New Mexico. The substructuring of genetic variation within the species complex was consistent with its division into two varieties, with genetic clusters within varieties generally associated with latitudinal zones. The analyses indicate widespread gene flow and/or recent common ancestry among genetic clusters within varieties, but not between varieties. Isolated disjunct populations had lower genetic variation by some measures and greater genetic differentiation than main-range populations. These results should be useful for decision-making and conservation planning related to this widespread and important species.