Salmonid fishes are cold water piscivores with a native distribution spanning nearly the entire temperate and subarctic northern hemisphere. Trout in the genus Oncorhynchus are the most widespread salmonid fishes and are among the most important fish species in the world, due to their extensive use in aquaculture and valuable fisheries. Trout that inhabit northwestern Mexico are the southernmost native salmonid populations in the world, and the least studied in North America. They are unfortunately also facing threats to their continued existence. Previous work has described one endemic species, the Mexican golden trout (O. chrysogaster), and one endemic subspecies, Nelson's trout (O. mykiss nelsoni), in Mexico, but previous work indicated that there is vastly more biodiversity in this group than formally described. Here we conducted a comprehensive genetic analysis of this important group of fishes using novel genetic markers and techniques to elucidate the biodiversity of trout inhabiting northwestern Mexico, examine genetic population structure of Mexican trout and their relationships to other species of Pacific trout, and measure introgression from non-native hatchery rainbow trout. We confirmed substantial genetic diversity and extremely strong genetic differentiation present in the Mexican trout complex, not only between basins but also between some locations within basins, with at least four species-level taxa present. We also revealed significant divergence between Mexican trout and other trout species and found that introgression from non-native rainbow trout is present but limited, and that the genetic integrity of native trout is still maintained in most locations. This information will help to guide effective conservation strategies for this important group of fishes.