Models of speciation by sexual selection propose that male-female coevolution leads to the rapid evolution of behavioral reproductive isolation. Here, we compare the strength of behavioral isolation to ecological isolation, gametic incompatibility, and hybrid inviability in a group of dichromatic stream fishes. In addition, we examine whether any of these individual barriers, or a combined measure of total isolation, is predicted by body shape differences, male color differences, environmental differences, or genetic distance. Behavioral isolation reaches the highest values of any barrier and is significantly greater than ecological isolation. No individual reproductive barrier is associated with any of the predictor variables. However, marginally significant relationships between male color and body shape differences with ecological and behavioral isolation are discussed. Differences in male color and body shape predict total reproductive isolation between species; hierarchical partitioning of these two variables’ effects suggests a stronger role for male color differences. Together, these results suggest an important role for divergent sexual selection in darter speciation but raise new questions about the mechanisms of sexual selection at play and the role of male nuptial ornaments.