The extent of inbreeding and outbreeding depression is thought to be a fundamental factor influencing the long term persistence of an endangered species. However, there have been no comprehensive examinations of either factor in an endangered species, except for a mouse species. Here we examined both the extent of inbreeding and outbreeding depression for five traits related to fitness in populations from the four watersheds in which the endangered Gila topminnow (Poeciliopsis o. occidentalis) still exists in the USA by making experimental matings with replicate samples and simultaneous controls. There was generally high survival, similar body size, and little bilateral asymmetry for all the inbred and outbred matings for all populations, i.e. there was no evidence of either inbreeding or outbreeding depression for these traits. Similarly there was no evidence of inbreeding or outbreeding effects for fecundity or sex ratio except for the sample from Monkey Spring, which had highly skewed (female-biased) sex ratios after one generation of inbreeding. In addition, brood sizes for Sharp Spring declined over time in inbred lines maintained by full-sib mating. These results further support the suggestion, based on molecular genetic analysis and considerations of habitat differences, that Monkey Spring be considered an evolutionarily significant unit, distinct from the remainder of existing populations. Potential explanations for the results and their implications for conservation are discussed.