Laos is the only landlocked country in Southeast Asia and borders Thailand, Myanmar and Cambodia, the three countries in this region that have been hardest hit by human immunodeficiency virus (HIV). Laos has been regarded as a low-HIV-prevalence country for decades. To understand the status of HIV in Laos in recent years, a retrospective study was performed among 2851 patients visiting a hospital in Vientiane, the capital of Laos, from November 2011 to May 2012. Whole blood samples were obtained from the patients, and DNA was extracted. HIV status was determined by HIV gag fragment-specific PCR assay. Sixty-nine samples were detected as HIV proviral DNA positive with a positive rate of 2.4% (69/2851). Sixty-one near full-length genomic sequences were obtained from the positive samples. The results of phylogenetic analysis showed that the vast majority (91.8%) of the HIV strains belonged to CRF01_AE, and the other five (8.2%) strains were identified as a new HIV circulating recombinant form CRF97_01B, which had a CRF01_AE backbone with an insertion of subtype B in the gag-pol region. Phylogeographic analysis revealed that HIV CRF01_AE circulating in Laos were multiply introduced from Thailand. These results indicated that Laos might be suffering a considerably more serious impact of HIV than previously believed. To keep this country from undergoing the same increase in HIV prevalence observed in its neighbors, immediate intervention measures and sufficient epidemiological research are urgently needed.