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Background: Trichomonas vaginalis (TV) infection, which is associated with poor reproductive tract health outcomes, is the most common curable nonviral STI globally with increasing evidence of treatment failure. TV epidemiology can be highly sub-structured and linked to low socio-economic status, older age in women and female sex work. However, the large highly repetitive genome makes whole genome sequencing approaches to describe TV transmission challenging. We use nested-PCR multi-locus sequence typing (MLST) of TV directly from clinical samples to describe strain diversity among two different groups of at-risk female sexworkers (FSWs) in Ecuador. Methods: DNA was extracted from self-collected vaginal swab samples from two groups of consenting FSWs: street-based workers (SBWs) and brothelbased workers (BBWs). Behavioural and demographic data were collected. Extracts were tested for TV using real-time PCR, TV-positive extracts amplified using a previously published twostep nested PCR for seven TV genes and MLSTamplicon libraries sequenced using Illumina MiSeq. Following alignment to reference genes, strain types were assessed using MLST software and a partition model phylogenetic tree built using IQTREE. Results: Among 250 FSWs, 35/205 (17%) and 23/45 (51%) BBWs and SBWs respectively were positive for TV (p < 0.001). A subset of 35 TV-positive vaginal DNA extracts were sequenced, of which 13/35 were SBWs and 22/35 BBWs. Compared with BBWs, SBWs were older (median age 42 vs 28; p < 0.001), earnt less (median 60US$ (IQR;45-125) vs 200US$ (IQR;100-300)), had higher rates of partner turnover and were more likely to work in one location. MLST typing appeared to show considerable numbers of possible new alleles. Sequences separated into one major (A) and three minor clades (B). All SBW sequences were clade A whereas clades A and B were found among BBWs. The possible influence of mixed infections in these 'high TV exposure' FSWs is under investigation. Conclusion: This study of TV strain diversity among FSWs in north-west Ecuador suggests a high level of allelic diversity. There appeared to be complete clade homogeneity among the poorer, single location, high client-turnover SBWs, compared to BBWs. TV-typing methods may be useful for identification of population structure in TV infection. [ABSTRACT FROM AUTHOR]