We examined barriers to gene flow in a hybrid zone of two subspecies of the song sparrow (Melospiza melodia). We focused on how mating signals and mate choice changed along an environmental gradient and gathered data on the morphology, genetics, ecology, and behavior across the zone. Melospiza m. heermanni of the Pacific slope of California and M. m. fallax of the Sonoran Desert, each distinct in plumage, meet across a steep environmental gradient in southeastern California. Although both subspecies occur in riparian habitat, their occupied habitat differs structurally, the former subspecies occurring in areas with denser understory and greater vertical heterogeneity. Song elements varied concomitantly, as predicted by the acoustic adaptation hypothesis, with heermanni having lower-pitched, more widely spaced elements. Females of both subspecies responded more strongly to homotypic than heterotypic song, and addition of subspecific plumage cues increased response if song was homotypic but not if heterotypic. Females thus assess multiple male traits, weighing song more heavily. Males of both subspecies showed significantly greater agonistic response to homotypic song. Microsatellite variation is correlated significantly with plumage variation across the zone and suggests limited gene flow between the taxa. The association of song and plumage with the environment and in turn with assortative mating suggests a means by which reproductive isolation may evolve or be maintained in hybrid zones.