The 2016 Conference on Retroviruses and Opportunistic Infections (CROI) highlighted hot spots in HIV infection. Men who have sex with men (MSM), transgender populations, people who inject drugs, fisherfolk, migrants, adolescents, and older adults are heavily impacted in a number of regions. Stigma contributes to risk behaviors and HIV acquisition across populations. HIV testing is a crucial first step in the HIV care continuum, and several large community-based surveys are underway in Africa to increase HIV testing, linkage to care, and uptake of antiretroviral treatment. Advances in preexposure prophylaxis (PrEP) featured prominently at CROI 2016. Two large efficacy trials of a vaginal ring containing the investigational drug dapivirine demonstrated efficacy and safety in preventing HIV infections in women in Africa. Data on the safety of long-acting injectable PrEP and several investigational PrEP drugs and formulations were also presented. Knowledge and use of PrEP among MSM in the United States appears to be increasing, and high uptake was seen among black MSM when provided as part of a culturally tailored support program. The use of broadly neutralizing antibodies for HIV prevention is a novel and promising approach to be evaluated in efficacy trials.