Objective: The purpose of this study was to assess the prevalence of sexual dysfunction (SD) and associated risk factors among middle-aged Latin American women using one validated instrument. Methods: The Female Sexual Function Index (FSFI) was applied to 7,243 healthy women aged 40 to 59 years who were users of 19 healthcare systems from 11 Latin American countries. An itemized questionnaire containing personal and partner sociodemographic data was also filled out. Results: Mean ± SD age of surveyed women was 49.0 ±5.7 years, with 11.6 years of schooling on average. There were 55.1% of women who were married, 46.8% who were postmenopausal, 14.1% who used hormonal therapy (HT), and 25.6% who were sexually inactive. Among those who were active (n = 5,391), the mean ± SD total FSFI score was 25.2 ± 5.9 and 56.8% of them presented SD (FSFI total score ≤26.55), with a prevalence varying from 21.0% to 98.5% depending on the center. Centers were grouped in terciles (according to mean ± SD prevalence). The tercile with higher SD prevalence (86.4%) compared with that with lower SD prevalence (32.2%) had significantly older women (49.5 ± 5.3 vs 48.0 ± 5.6 y) with a higher rate of vaginal dryness (60.4% vs 40.8%) and older partners (53.0 ± 6.9 vs 50.2 ± 7.5 y). Similarly, there was a significantly higher rate of married (68.5% vs 63.1%), postmenopausal (49.7% vs 39.3%), and HT-using women (23% vs 9.2%). There were no differences in regard to their health perception, history of oophorectomy, rape, and partner SD rate (27% vs 26.2%). The total FSFI score was significantly lower in the tercile with higher SD prevalence (22.0 ± 5.0 vs 27.5 ± 5.4). Logistic regression analysis was used to determine the odds ratios (95% CIs) for the main risk factors associated with SD among those who were sexually active: bad lubrication, 3.86 (3.37-4.43); use of alternative menopausal therapies, 2.13 (1.60-2.84); partner SD, 1.89 (1.63-2.20); older women (>48 y), 1.84 (1.61-2.09); bladder problems, 1.47 (1.28-1.69); HT use, 1.39 (1.15-1.68); negative perception of female health status, 1.31 (1.05-1.64); and being married, 1.22 (1.07-1.40). Protective factors were higher educational level (women), partner faithfulness, and access to private healthcare. Conclusions: The prevalence of SD in this middle-aged Latin American series was found to be high, varying widely in different populations. A decrease in vaginal lubrication was the most important associated risk factor. Differences in the prevalence of risk factors among the studied groups, several of which are modifiable, could explain the variation of SD prevalence observed in this study.