Objectives Despite a growing HIV threat, there is no definition and characterisation of key populations (KPs), who could be the major drivers of the epidemic in Turkey. We used programmatic mapping to identify locations where KPs congregate, estimate their numbers and understand their operational dynamics to develop appropriate HIV programme implementation strategies. Methods Female and transgender sex workers (FSWs and TGSWs), and men who have sex with men (MSM) were studied in Istanbul and Ankara. Within each district, hot spots were identified by interviewing key informants and a crude spot list in each district was developed. The spot validation process was led by KP members who facilitated spot access and interviews of KPs associated with that spot. Final estimates were derived by aggregating the estimated number of KPs at all spots, which was adjusted for the proportion of KPs who visit multiple spots, and for the proportion of KPs who do not visit spots. Results FSWs were the largest KP identified in Istanbul with an estimate of 30 447 (5.8/1000 women), followed by 15 780 TGSWs (2.9/1000 men) and 11 656 MSM (2.1/1000). The corresponding numbers in Ankara were 9945 FSWs (5.2/1000 women), 1770 TGSWs (1/1000 men) and 5018 MSM (2.5/1000 men). Each KP had unique typologies based on the way they find and interact with sex partners. MSM were mostly hidden and a higher proportion operated through internet and phone-based applications. Night time was the peak time with Friday, Saturday and Sunday being the peak days of activity in both Istanbul and Ankara. Conclusions This study has highlighted the presence of a substantial number of FSWs, TGSW and MSM in Istanbul and Ankara. The information obtained from this study can be used to set priorities for resource allocation and provide HIV prevention services where coverage could be the highest.