Neisseria gonorrhoeae penicillinase-producing (PPNG) plasmid is responsible for dissemination of high-level penicillin resistance among gonococci worldwide. The first PPNG plasmid was isolated in South-East Asia, named the ‘Asian’ plasmid, and is 7426 base-pair (bp) in size. Further variants of the ‘Asian’ plasmid have since been described and named on the basis of their isolation origin; the ‘African’ (5599 bp), ‘Rio-de-Janerio/Toronto’ (5154 bp), ‘Nimes’ (6798 bp), ‘New-Zealand’ (9309 bp) and ‘Johannesburg’ (4865 bp) plasmids. This sub-study was conducted as part of a NHMRC-funded study titled GRAND (Gonorrhoea Resistance Assessment via Nucleic acid Detection). This study examined the conservation of sequence targets utilised by a previously described PPNG real-time PCR assay and used to detect PPNG strains in non-cultured but PCR positive clinical samples. The assay acts as an indirect marker of penicillinase activity, by amplifying a region of sequence predicted to be conserved across all known PPNG plasmid types, while not specifically targeting the penicillinase (bla) coding sequence. This study used the PPNG-PCR to screen 351 PPNG isolates from reference laboratories throughout Australia for the first six months of 2012 which comprised approximately 98% of all PPNG strains isolated in Australia in this time period. In total 350/351 (99.7%) of isolates provided positive results by the PPNG-PCR, however one isolate from South Australia provided negative results. Complete DNA sequencing of this plasmid sequence revealed a plasmid of 3269 bp, the smallest such gonococcal plasmids described to date. Notably, the plasmid exhibited a previously undescribed 1885bp deletion which omitted the PPNG-PCR oligonucleotide targets.While the prevalence of gonococci carrying this plasmid in our population is suggested to be very low, the failure to detect this novel ‘Australian’ plasmid by the PPNG-PCR further highlights the challenges of using molecular methods for N. gonorrhoeae diagnostics and characterisation.