Background: The accurate laboratory identification of Neisseria gonorrhoeae is an essential element of the diagnosis of gonorrhoea and is particularly important for medicolegal cases. The detection of proline iminopeptidase (Pip) activity is widely used as a marker for gonococci, although Pip negative N gonorrhoeae isolates have been shown to generate false negative identifications when using biochemical kits. This study aimed to determine the frequency of Pip negative gonococci in England and Wales. Methods: A total of 2055 isolates were collected from consecutive patients attending 26 genitourinary medicine centres as part of the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP). Upon receipt the identity of all isolates was confirmed using N gonorrhoeae specific monoclonal antibodies and the Pip status was determined using the Gonochek II kit. Results: The overall prevalence of Pip negative isolates was found to be 4.33%. Significant geographical variation was observed between isolates from centres outside London (p ≤ 0.001). Variation was also observed within London between the nine different clinics submitting isolates (p = 0.025). There was also a higher frequency of these isolates among men who have sex with men (p ≤ 0.001), which may account for geographical variations. Conclusion: Pip negative N gonorrhoeae isolates are a very serious cause for concern as currently all biochemical test kits available within the United Kingdom require the presence of the Pip enzyme for an unambiguous identification of this pathogen. Raising awareness of the current prevalence of Pip negative N gonorrhoeae isolates is critical for the successful control of gonorrhoea.