Sequences from the mitochondrial cytochrome b gene were used to examine patterns of variation within and among populations of the ferruginous pygmy-owl (Glaucidium brasilianum) from both North America (including populations from Mexico) and South America. As currently conceived, G. brasilianum is paraphyletic, with North American and South American clades representing two distinct groups that should be recognized as the distinct species G. ridgwayi and G. brasilianum, respectively. Within the G. ridgwayi clade, populations from Arizona, Sonora, and Sinaloa are genetically distinct and share no mitochondrial haplotypes with populations occurring in Texas and other regions of Mexico. According to nested clade analysis this separation may be the consequence of past fragmentation that predates the origin of the Sonora desert. In addition, gene flow between the Arizona/Sonora/Sinaloa populations and the Texas/other Mexico populations is practically nonexistent, with estimates being approximately one individual every 10 generations. Collectively, these data suggest that the Arizona/Sonora/Sinaloa clade should be recognized as either a distinct subspecies or phylospecies from the group containing populations in Texas and the remainder of Mexico. These data should be used as guidelines for pygmy-owl recovery and conservation, as they meet the recommendations of task 3 of the pygmy-owl recovery plan that lists genetic data as essential information for pygmy-owl management.