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BACKGROUND Female sex workers (FSW), men who have sex with men (MSM), and transgender women (TGW) are at high risk of acquiring HIV in many settings, such as Papua New Guinea (PNG). An understanding of the approximate size of these populations can inform resource allocation for HIV services for FSW, MSM, and TGW. OBJECTIVE An objective of this multi-site survey was to conduct updated population size estimations (PSE) of FSW and MSM/TGW. METHODS Respondent-driven sampling (RDS) biobehavioral surveys of FSW and MSM/TGW were conducted in 3 major cities—(1) Port Moresby, (2) Lae, and (3) Mount Hagen—between June 2016 and December 2017. Eligibility criteria for FSW included: (1) ≥12 years of age, (2) born female, (3) could speak English or Tok Pisin (PNG Pidgin), and (4) had sold or exchanged sex with a man in the past six months. Eligibility for MSM/TGW included: (1) ≥12 years of age, (2) born male, (3) could speak English, or Tok Pisin, and (4) had engaged in oral or anal sex with another person born male in the past six months. PSE methods included unique object multiplier, service multiplier, and successive sampling-population size estimation (SS-PSE) using imputed visibility. Weighted data analyses were conducted using RDS-Analyst and Microsoft Excel. RESULTS Sample sizes for FSW and MSM/TGW in Port Moresby, Lae, and Mount Hagen included: (1) 673 and 400, (2) 709 and 352, and (3) 709 and 111 respectively. Keychains were used for the unique object multiplier method and were distributed 1 week before the start of each RDS survey. HIV service testing data were only available in Port Moresby and Mount Hagen and SS-PSE estimates were calculated for all cities. Due to limited service provider data and uncertain prior size estimation knowledge, unique object multiplier weighted estimations were chosen for estimates. In Port Moresby, we estimate that there are 16,053 (95% CI 8232-23,874) FSW and 7487 (95% CI 3975-11,000) MSM/TGW, approximately 9.5% and 3.8% of the female and male populations respectively. In Lae, we estimate that there are 6105 (95% CI 4459-7752) FSW and 4669 (95% CI 3068-6271) MSM/TGW, approximately 14.4% and 10.1% of the female and male populations respectively. In Mount Hagen, we estimate that there are 2646 (95% CI 1655-3638) FSW and 1095 (95% CI 913-1151) MSM/TGW using service multiplier and successive sampling, respectively. This is approximately 17.1% and 6.3% of the female and male populations respectively. CONCLUSIONS As the HIV epidemic in PNG rapidly evolves among key populations, PSE should be repeated to produce current estimates for timely comparison and future trend analysis.