Use of HIV prevention methods may vary for women by types of sexual partners. In a microbicide safety and effectiveness trial (HPTN 035) differences in adherence to a microbicide study gel were compared between women with new versus ongoing partnerships over time. 1,757 women in the three HPTN 035 trial’s arms completed the Follow-up Partner Status (FPS) questionnaire at their last study visit. Women married at baseline were asked if they had the same husband, new husband or new partner. Unmarried women were asked if they had changed partners or married. Self-reported gel adherence during the last sex act was compared at each quarterly visit between women with ongoing versus new partners. High gel adherence was compared with low gel adherence (85–100 vs. <85 % of last vaginal sex acts reported with gel use, respectively) in multivariable models to assess associations with partner change. Overall 7 % of women (n = 123) reported a new partner and 41 % (51) of those reported a new husband. Median gel adherence was reported to be 100 % in women with ongoing partners and 75 % for women with new partners (p < 0.001). In women reporting no gel use in their last sex act, only 12.5 % of the women with a new partner and none of those with an ongoing partner reported using condoms (p < 0.001). Fewer women with new partners reported using both the gel and condom during the last sex act as compared to women with ongoing partners (median 50 vs. 71.4 %, p < 0.001). After adjusting for age, site, education level, and sexual frequency, women with ongoing partners were more likely to report high gel adherence than those with new partners (AOR 2.5, 95 % CI 1.6, 3.9). This pattern persisted when gel use over time was compared between women with new versus ongoing partners. In the HPTN 035 trial, women with new partners had higher HIV incidence and reported less gel use and higher condom use. Specific counseling and support are needed to help women use potential HIV prevention methods, including microbicides, when they are changing partners.