Female sex hormones are known to regulate the immune functions of the female genital tract (FGT). While the majority of immune cells found in the FGT are T cells and Natural Killer cells (NK cells), very few studies have focused on NK cells in the FGT in contrast to numerous studies focusing on T-cells. Here, we characterized the distribution, phenotype and function of NK cells in FGT of HIV seronegative women using different mucosal sampling methods; cervicovaginal lavage (CVL), endocervical cytobrush (CB), and cervicovaginal biopsy and compared them to blood. We isolated cells from blood and FGT and performed multi-color flow cytometry to identify cellular phenotypes. First, we looked at the distribution of CD56brightand CD56dimpopulations of NK cells across the samples and found that CD56brightNK cells were lower in the FGT tissues compared to blood. Between the CD56brightand CD56dimpopulations, the CD56dimpopulation was significantly reduced in the follicular phase compared to the luteal phase in tissues. The NK cells within the FGT samples express higher levels of tissue resident markers CD69 and CD103. The co-expression of CD69+CD103+ markers seem to be significantly higher in follicular phase compared to luteal phase in tissues. These cells have high proliferative capacity with high levels of HLADR expression, suggesting that these cells are highly functional and activated. The gut tissue homing marker alpha4beta7 expression significantly reduced in the follicular phase, however other homing markers CCR5 and CCR7 expression were elevated in the follicular phase. Altogether, these data demonstrate that FGT is enriched with tissue resident NK cells with high activation and proliferation profile with distinct homing potential.