In a study conducted over 30 years ago, a remarkable population of amphipod crustaceans ( Gammarus minus) was discovered in which 100% of the females displayed intersex characteristics. Such high levels of intersexuality are atypical amongst gonochoristic crustaceans and current theories relating to sex allocation suggest intermediate (intersex) forms should be competitively selected against. This study set out to confirm the existence of this extraordinary population and to compare it with other G. minus populations in various spring and cave systems across Pennsylvania, Virginia and West Virginia (USA), with varying environmental characteristics in an attempt to elucidate the prevalence and cause of intersexuality in this species. Results from this study verify the continued existence of these highly abundant, intersex female populations of G. minus, with proportions ranging between 73% and 100%. These populations were also associated with highly male-biased sex ratios, which is equally unusual amongst gonochoristic crustaceans. Intersex female specimens had a reduced fecundity compared to normal females, although this result was not statistically significant. Although no feminising microsporidian parasites were observed through histological examination, the presence of a novel feminising parasitic factor cannot be ruled out. The continued vigour of these populations over three decades suggests that anthropomorphic factors are not causing the high levels of intersexuality observed. Intersexuality was absent or very low in frequency (<1%) in most other springs that we investigated. Therefore, G. minus appears to represent an interesting and novel model system for studying sex determination.