Objective: To obtain HIV incidence among injection drug users (IDU) and female sex workers (FSW) in Dehong Prefecture, Yunnan Province during 2009-2017. Methods: We recruited drug users and female sex workers from all sentinel surveillance sites across Dehong Prefecture during 2009-2017. A total of 10 480 IDU and 18 126 FSW in Dehong Prefecture were recruited by fingerprint technique. Data about drug uses, commercial sexual behavior, sociodemographic characteristics was collected by structured questionnaire. HIV-positive patients who were long-term infected or with CD4⁺ T cell count was ≤200 were not included for further HIV incidence testing. Also, those who self-identified as on antiretroviral treatment (ART) or AIDS cases were also excluded. A total of 841 and 157 plasma specimens from IDU and FSW that met the inclusion criterion were finally included, respectively. Limiting antigen avidity enzyme immunoassay(LAg-Avidity EIA) were performed to calculate the HIV incidence among these two sub-populations. Results: A total of 3 444 IDU were HIV-positive, among which 884 (25.7%) were Burmese with age of (30.4±7.7), and 2 560 were Chinese with age of (36.6±7.3). Among 228 HIV-positive FSW, 109 (47.8%) were Burmese with age of (27.1±6.3), 119 (52.5%) were Chinese with age of (29.9±11.1). For IDU, the estimated HIV incidence among Burmese in 2009-2010, 2011-2012, 2013-2014, 2015-2017 was 4.20% (95%CI: -0.55%-8.95%), 7.75% (95%CI: 2.95%-12.55%), 11.79% (95%CI: 5.38%-18.20%), 10.30% (95%CI: 5.67%-14.94%), respectively, while Chinese were 3.11% (95%CI: 1.59%-4.64%), 0.03% (95%CI: -0.03%-0.08%), 1.55% (95%CI: 0.54%-2.57%), 0.58% (95%CI: -0.06%-1.04%), respectively. In 2009-2011, 2012-2014, 2015-2017, estimated HIV incidence among Burmese FSW was 0.22% (95%CI: -0.21%-0.64%), 1.24%(95%CI: 0.15%-2.32%), 0.55%(95%CI: 0.01%-1.08%). Whereas, estimated HIV incidence among Chinese FSW was 0.62% (95%CI: 0.25%-0.98%), 0.11% (95%CI: -0.04%-0.26%), 0.22% (95%CI: 0-0.44%). Conclusion HIV incidences among Chinese IDU and FSW are on the downward trend, while Burmese IDU and FSW seem to be gaining momentum.