Neisseria gonorrhoeae is a public health concern due to increasing numbers of cases of gonorrhoea and the ability of the organism to develop antimicrobial resistance. N. gonorrhoeae can harbour β-lactamase plasmids that encode TEM-1 penicillinase, but do not produce extended spectrum β-lactamases (ESBLs). ESBLs are active against the last remaining option for gonorrhoea monotherapy, ceftriaxone. The aim of this research was to establish what resistance mechanisms in N. gonorrhoeae could abolish the effectiveness of ceftriaxone for the treatment of gonorrhoea. Investigations into the genetic diversity of blaTEM alleles in gonococcal isolates from 2012 detected a high proportion of blaTEM-135 alleles (27%). Only a single specific mutation near the β-lactamase active site could result in TEM-135 penicillinase evolving into an ESBL. Electroporation was established for the transformation of native gonococcal resistance plasmids into N. gonorrhoeae, and was then used in attempts to transfer the enteric plasmid pEK204 (harbouring blaCTX-M-3 and blaTEM-1) into gonococcal strains. Electroporation and natural transformation were additionally used to transform gonococci with blaCTX-M-3 and blaTEM-10 genes. Five transformants were detected using blaTEM-10 and these all showed increased minimum inhibitory concentrations of ceftriaxone. The lack of success in uptake of pEK204 and blaCTX-M-3 was probably due to large plasmid size and lack of recombination site, respectively. Nevertheless, gonococcal β-lactamase plasmids were successfully transferred into clinical strains of the multidrug-resistant clone N. gonorrhoeae ST1407, suggesting that this could also happen in natural mixed gonococcal infections. In summary, it is encouraging that no further blaTEM alleles were detected and that N. gonorrhoeae was not able to express a CTX-M-type ESBL. However, the expression of TEM-10 ESBL is concerning and this work is the first report of ESBL activity in gonococci, albeit in vitro. It is essential to continue antimicrobial susceptibility surveillance and to develop molecular surveillance to detect rapidly the emergence of an ESBL in N. gonorrhoeae.