CD4⁺ T follicular helper cells (T_FH) in germinal centers are required for maturation of B-cells. While the role of T_FH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of T_FH (CXCR5⁺PD-1⁺⁺) and precursor-T_FH (CXCR5⁺PD-1⁺) cells. The majority of T_FH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of T_FH-cells mainly in the Peyer’s patches and FRT. The novel finding of T_FH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study T_FH-cells and to evaluate HIV-1 vaccines.