Immediate antiretroviral therapy (ART) for acute HIV infection (AHI) may decrease HIV transmission in high-risk populations. This study evaluated knowledge of AHI and AHI testing program preferences in Lima, Peru through four semi-structured focus groups with high-risk men who have sex with men (MSM) (n = 20) and transgender women (TW) (n = 16). Using content analysis, emergent themes included knowledge of AHI symptoms, AHI transmission potential, and the HIV testing window period, and preferences concerning point of care results. Participants demonstrated low familiarity with the term AHI, but many correctly identified AHI symptoms. However, these symptoms may not motivate testing because they overlap with common viral illnesses and AIDS. Some were aware that infectiousness is highest during AHI, and believe this knowledge would facilitate HIV testing. The shortened window period with AHI testing would encourage testing following high-risk sex. Delayed result notification would not decrease AHI testing demand among MSM, although it might for some TW.