In Iran, People Who Inject Drugs (PWID), Female Sex Workers (FSW), and prisoners are the main key populations at risk of HIV infection. This study aimed to evaluate the trend of HIV incidence among PWID, FSW and prisoners as an impact measure of HIV harm reduction and prevention efforts in Iran. Data were obtained from the two rounds of national bio-behavioral surveillance surveys among FSW (2010 (n = 872), 2015 (n = 1339)), PWID (2010 (n = 2417), 2014 (n = 2307)), and prisoners (2009 (n = 4536), 2013 (n = 5390)) through facility-based (FSW and PWID surveys) and cluster sampling (prisoner surveys). Time-at-risk was calculated assuming the age at first sex or drug injection as the beginning of the at-risk period and the age at the time of the interview or date when they received a positive HIV test result as the end of this period, adjusted for interval censoring. HIV incidence among PWID in 2014 was 5.39 (95% CI 4.71, 6.16) per 1,000 person-years (PY), significantly lower than in 2009 (17.07, 95% CI 15.34, 19.34). Similarly, HIV incidence was 1.12 (95% CI 0.77, 1.64) per 1,000 PY among FSW in 2015, a significant drop from 2010 (2.38, 95% CI 1.66, 3.40). Also, HIV incidence decreased among prisoners from 1.34 (95% CI: 1.08, 1.67) in 2009 to 0.49 (95% CI: 0.39, 0.61) per 1,000 PY in 2013. Our findings suggest that after an increase in the 2000s, the HIV incidence may have been decreased and stabilized among key populations in Iran.