Summary Using the twoline pupfish (Cyprinodon bifasciatus), a species with a resource-based polygynous breeding system, we examined male mating success in the wild, and we experimentally investigated effects of male body size and substrate type on female association patterns in the laboratory. Our purpose was to (a) identify the traits contributing to male reproductive success in the field, (b) measure preferences for each trait independently in the laboratory, and (c) determine the relative importance of each trait. Field observations revealed that substrate type was the main determinant of male reproductive success: males defending territories on rocks mated significantly more often than males defending territories on silt or sand. Laboratory experiments supported the field data, and revealed that the female preference for substrate type was independent of male body size effects. When given a choice between two males matched for size but differing in the type of substrates they were defending, females preferred the male on the rocky substrate over the male on the sandy substrate. Laboratory experiments also revealed a female preference for larger males when substrate type was held constant. Finally, when females were presented with a choice between a large male on a sandy substrate and a small male on a rocky substrate, no clear preference emerged. We provide several interpretations for this result, and we argue that both traits may be strong predictors of the male's competitive ability.