Xishuangbanna is a popular tourist destination in southern Yunnan Province, bordering with Vietnam and Laos, and Myanmar. There are more than 2 million cross-border travelers annually entering China at two land ports, Daluo and Mohan. To clarify the characteristics of HIV-1 prevalence among entry travelers, a retrospective epidemic investigation was conducted. Between 2003 and 2012, 196 among 91 432 entry travelers were detected to be HIV-1-positive, accounting for infection rate of 0.21%. Eighty-one gag-pol gene sequences were obtained for phylogenetic analyses and subsequent recombination analysis. It was shown that the most prevalent HIV-1 subtype in this population was circulating recombinant form (CRF) 01_AE (42, 51.86%), followed by CRF08_BC (17, 20.99%), CRF07_BC (4, 4.94%), B' (2, 2.47%), C (2, 2.47%), other recombinants (9, 11.11%), and undefined subtype (5, 6.17%). HIV-1 genotype distribution among travelers entering at two land ports is different, CRF01_AE and CRF08_BC accounted for a larger percentage among individuals from Daluo, whereas the proportions of subtype C and undefined URFs were larger among individuals from Mohan. The undefined subtype indicates the occurrence of novel HIV-1 subtype or CRF. This finding is useful for developing of AIDS prevention strategies.