Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach and this can result in a reduced breeding capability with potential population level impacts. Complete male to female sex reversal in roach has been induced experimentally for exposures to WwTW effluents and a steroidal oestrogen, but whether this occurs in wild populations cannot be assessed in the absence of genetic sex probe. Through restriction site-associated DNA sequencing (RAD-seq), we isolated a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from non-polluted rivers. We then applied the genetic sex marker to a series of samples from exposures to oestrogen/oestrogenic effluents exposures to confirm sex reversal in those exposures from males to females, to confirm unequivocally that intersex results from feminisation of males in wild populations, and show no evidence for complete sex reversal in wild roach sampled from WwTW effluent contaminated river sites. We furthermore, confirm that sex reversed males are able to breed as females.