Background Women who inject drugs (WID) face multifaceted challenges and remain one of the most invisible and vulnerable HIV-impacted populations, disproportionately affected by stigma, health disparities, and structural inequalities. Accurate population size estimation and analysis of the HIV care continuum among this group are crucial for effective programmatic planning. This study aims to describe the main characteristics of the WID participants in the southern region of Mozambique, analyze the self-reported progress towards the 2nd and 3rd targets of the 95-95- 95 framework (ART uptake and viral suppression), and estimate the size of this population. Methods We conducted a cross-sectional study using a three-source capture-recapture (3 S-CRC) method to estimate the population size of WID in the southern provinces of Mozambique, covering Maputo City, Maputo Province, Gaza, and Inhambane. A separate structured survey was administered during each round to gather sociodemographic and HIV-related information, and the survey results from the first encounter of each participant were used for descriptive results to avoid double or triple reporting from recapture encounters. The HIV care cascade was analyzed, focusing on the second 95% (those aware of their HIV status and currently on antiretroviral therapy (ART) and the third 95% (viral suppression among those on ART), based on self-reported status. Non-parametric Bayesian modeling was applied for estimation, based on the 2022 country population projections. Data were analyzed using R software. Results A total of 159 WID were enrolled in Maputo City, 239 in Maputo Province, 29 in Gaza, and 168 in Inhambane. The majority in Maputo City (54.7%), Maputo Province (52.7%), and Inhambane (73.2%) were aged 25–31. Nearly 90% of WID reported engaging in sex work. The HIV care continuum analysis revealed significant gaps in ART uptake and adherence, with Maputo City and Inhambane in particular falling well below the UNAIDS 95-95-95 targets, at 61.0% and 78.9% of self-reported HIV-positive WID reporting current ART treatment, respectively. In all provinces, selfreported viral suppression rates were below the 95% target. The median population size estimation was 240 (173–411; 0.05–0.13% of the adult female population aged 18–49) for Maputo City, 1160 (557-2491; 0.08–0.37%) for Maputo Province, 40 (29–78; 0.01–0.02%) for Gaza, and 650 (381-1083; 0.09–0.27%) for Inhambane. Conclusion This study provides the first population size estimates for WID in Mozambique and identifies critical gaps in the HIV care continuum. The insights gained underscore the urgent need for focused, comprehensive health services to address the complex needs of WID and inform public health planning. Improving efforts to meet global HIV targets and enhance health outcomes