Background: The prevalence of the sexually transmissible protozoan parasite Trichomonas vaginalis in the highlands of Papua New Guinea (PNG) has been reported to be as high as 46% and although not previously studied in Papua New Guinea, clinical resistance against metronidazole (Mz), the drug most commonly used to treat trichomoniasis, is well documented worldwide. This study was primarily aimed at assessing resistance to Mz in T. vaginalis strains from the Goroka region. Methods: Consenting patients presenting at the Goroka Base Hospital Sexually Transmitted Diseases (STD) Clinic and local women were asked to provide two vaginal swabs: one for culturing of the parasite; and one for polymerase chain reaction detection of T. vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae. T. vaginalis isolates were assayed for Mz susceptibility and a selection was genotyped. Results: The prevalence of T. vaginalis was determined to be 32.9% by culture and polymerase chain reaction of swabs among 82 local women and patients from the STD clinic. An unexpectedly high level of in vitro Mz resistance was determined with 17.4% of isolates displaying unexpectedly high resistance to Mz. The ability to identify isolates of T. vaginalis by genotyping was confirmed and the results revealed a more homogeneous T. vaginalis population in Papua New Guinea compared with isolates from elsewhere. Conclusion: T. vaginalis is highly prevalent in the Goroka region and in vitro Mz resistance data suggest that clinical resistance may become an issue.