Previous laboratory based studies have shown that oil sands process-affected waters (OSPWs) containing high concentrations of naphthenic acids (>25 mg/l) have adverse effects on the reproductive physiology of fish. The purpose of this study was to assess the reproductive development and health of a wild population of fathead minnows (Pimephales promelas) inhabiting an OSPW pond that has moderate concentrations of naphthenic acids (∼10 mg/l). Fathead minnows were collected at various times during the period of 2006 through 2008 from Demonstration Pond (OSPW) located at Syncrude Canada Ltd., and two reference sites, Beaver Creek reservoir and Poplar Creek reservoir, which are all north of Fort McMurray, AB, Canada. Condition factor, gill histopathology, gonadosomatic indices, liver somatic indices, male secondary sexual characteristics, and plasma sex steroids were examined. Depending on the time of year that fathead minnows were collected, there were differences in the condition factor, gonadosomatic indices, liver somatic indices, and secondary sexual characteristics of fathead minnows (in males) from Demonstration Pond when compared to the fathead minnows from the reference sites. In comparison to reference fish, lower concentrations of 11-ketotestosterone were measured in the plasma of male fathead minnows collected from Demonstration Pond in June 2006 and July 2007. Black spot disease and Ligula intestinalis were prevalent in fathead minnows from the reference sites but were not observed in fathead minnows from Demonstration Pond. The opercula of fathead minnows from Demonstration Pond also differed from those of reference fish. An examination of the gills of fathead minnows from Demonstration Pond revealed that were a number of proliferative and degenerative alterations relative to reference fish. Even though the fathead minnow population has been maintained in this OSPW pond since 1993, the results of this study demonstrate that the OSPW continues to affect the reproductive development and health of the fathead minnows compared to fish collected at reference sites.