The intertidal amphipod (Echinogammarus marinus LEACH) exhibits several forms of intersexuality (intersex males and intersex females) and has been considered an ideal model to study reproductive endocrine disruption in Crustacea. This study aimed to investigate both the physiology and gene expression in intersex E. marinus with the objective of better understanding their reproductive biology and aid the development of biomarkers of de-masculinisation in Crustacea. E. marinus populations from three Scottish (Thurso, Inverkeithing and Loch Fleet) and two English sites (Portsmouth Harbour and Langstone Harbour) were assessed for intersexuality prevalence, sperm counts or microsporidian infection rate. Increased incidence of intersexes and reduced sperm counts were revealed in amphipods from industrial impacted sites. The microsporidian species infecting E. marinus were identified for the first time, and Dictyocoela duebenum and Dictyocoela berillonum were found to be dominant in Inverkeithing and Portsmouth Harbour E. marinus populations, respectively. Microsporidian has been reported as a potential factor inducing intersexes in amphipods, and this study also revealed a strong association between microsporidian infection and intersexes in E. marinus. A cross-species cDNA microarray was used to characterise the gene expressions of three male phenotypes (normal males, internal and external intersex males), and PCA analysis clearly differentiates the three groups into three separate patches. The de novo transcriptome sequencing was carried out on E. marinus gonadal tissue, by employing Roche 454 pyrosequencing producing one of the largest cDNA libraries for a crustacean species. A total of 12,645 gonadal contigs were assembled from 213,212 sequencing reads, and 1206, 1745 and 782 contigs were found to be male-, female- and intersex-specific genes, respectively. This study revealed that the majority of internal intersex males were without microsporidian infection, indicating multiple factors causing intersexuality in E. marinus. Molecular studies identified differential gene expression patterns amongst sexual phenotypes and suggest the various forms of intersexuality in E. marinus are different at both the biological and genetic level. A large number of strongly male and female sex biased gene sequences have been identified which will provide a powerful resource for future studies into the reproductive biology of crustaceans.