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Objectives The aim of the study was to estimate the levels of transmitted drug resistance (TDR) in HIV-1 using very sensitive assays to detect minority drug-resistant populations. Methods We tested unlinked anonymous serum specimens from sexual health clinic attendees, who had not received an HIV diagnosis at the time of sampling, by both standard genotyping and using minority detection assays. Results By standard genotyping, 21 of 165 specimens (12.7%) showed evidence of drug resistance, while, using a combination of standard genotyping and minority mutation assays targeting three commonly observed drug resistance mutations which cause high-level resistance to commonly prescribed first-line antiretroviral therapy (ART), this rose to 32 of 165 (19.4%). This increase of 45% in drug resistance levels [95% confidence interval (CI) 15.2–83.7%; P=0.002] was statistically significant. Almost all of this increase was accounted for by additional detections of the M184V mutation. Conclusions Future surveillance studies of TDR in the United Kingdom should consider combining standard genotyping and minority-specific assays to provide more accurate estimates, particularly when using specimens collected from chronic HIV infections in which TDR variants may have declined to low levels.