Local adaptation is often invoked to explain hybrid zone structure, but empirical evidence of this is generally rare. Hybrid zones between two poeciliid fishes, Xiphophorus birchmanni and X. malinche, occur in multiple tributaries with independent replication of upstream-to-downstream gradients in morphology and allele frequencies. Ecological niche modelling revealed that temperature is a central predictive factor in the spatial distribution of pure parental species and their hybrids and explains spatial and temporal variation in the frequency of neutral genetic markers in hybrid populations. Among populations of parentals and hybrids, both thermal tolerance and heat-shock protein expression vary strongly, indicating that spatial and temporal structure is likely driven by adaptation to local thermal environments. Therefore, hybrid zone structure is strongly influenced by interspecific differences in physiological mechanisms for coping with the thermal environment.