Detection of people with acute HIV infection (AHI) affords an important opportunity for early HIV treatment and prevention. HIV RNA reverse transcriptase-polymerase chain reaction (RT-PCR) testing with two-stage pooling scheme was used to detect the AHI in specimens collected from sexually transmitted disease (STD) clinic patients in Guangxi, China. A total of 246 HIV RNA tests were required to screen 11 395 samples negative for conventional enzyme immunoassay (EIA) and Western blot assays, and five AHI cases (0.04%, 95%CI 0.02% to 0.10%) with a high viral load (median of 265,677 copies per ml) were detected. The total expenditure for RT-PCR testing reflected an added cost of $2.9 per specimen screened and $6575 per additional case of AHI identified among the study population. This study supports the feasibility of pooled RNA testing in addition to detection of HIV infections among patients at STD clinics in China, but the cost effectiveness should be carefully considered.