INTRODUCTION: Condomless anal intercourse (AI) confers a far greater likelihood of HIV transmission than condomless vaginal intercourse (VI). However, little is known about AI practice over the life course of women, to what extent AI practice is condom-protected, and whether it is associated with other HIV risk behaviors. We aim to describe longitudinal AI practice among HIV-seronegative women and to identify subgroups with distinct trajectories of AI practice. METHODS: Using data from the Women's Interagency HIV Study, an observational cohort of US women with or at risk for HIV, we described AI practice among HIV-seronegative participants. Group-based trajectory modeling was used to identify distinct AI trajectories. We used multinomial regression to examine associations between baseline characteristics and trajectory group membership. RESULTS: A third of the 1,085 women in our sample reported any AI over follow-up (median follow-up = 14 years). AI decreased more sharply with age compared to VI. Consistent condom use during AI was low: twice the proportion of women never reported using condoms consistently during AI compared to during VI. 5 trajectory groups were identified: AI & VI persistors (N = 75) practiced AI and VI consistently over follow-up (AI & VI desistors (N = 169) tended to practice AI and VI when young only, while VI persistors (N = 549), VI desistors (N = 167), and AI & VI inactives (N = 125) reported varying levels of VI practice, but little AI. AI & VI persistors reported multiple male partners and exchange sex at more visits than other groups. Women who identified as bisexual/lesbian (vs heterosexual), who had ever experienced physical and sexual violence (vs never), and/or who reported above the median number of lifetime male sex partners (vs median or below) had approximately twice the odds of being AI & VI persistors than being VI persistors. CONCLUSIONS: We identified a small subgroup of women who practice AI and report inconsistent condom use along with other risk behaviors throughout the life course; they may therefore particularly benefit from ongoing access to HIV prevention services including pre-exposure prophylaxis. Owen BN, Baggaley RF, Maheu-Giroux M, et al. Patterns and Trajectories of Anal Intercourse Practice Over the Life Course Among US Women at Risk of HIV. J Sex Med 2020;17:1629-1642.