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Background: Adherence to option B+ antiretroviral treatment (ART) is vital to a successful implementation of Prevention of Mother to Child Transmission (PMTCT) program. Further studies show that optimal viral suppression is also crucial for a successful PMTCT program, however barriers to adherence exist and differ among populations and particularly within few years of its adoption in Tanzania. This study therefore aimed at investigating the level and predictors of adherence to ART option B+ among pregnant and lactating women in rural and urban settings of eastern Tanzania. Methodology: A cross-sectional study was conducted among 305 pregnant women and lactating mothers on Option B+ regime from six health facilities located in rural and urban settings in Morogoro region in eastern Tanzania. Data were collected using a structured questionnaire. Data analysis was performed using descriptive statistics, as well as bivariate and multivariate logistic regression. Results: Good adherence to option B+ PMTCT drugs was 26.3% and 61.1% among respondents residing in urban and rural areas respectively. The rural residents were 4.86 times more likely to adhere compared to their counterparts in an urban area (aOR = 4.86; 95% CI = 2.91–8.13). Similarly, women with male partners’ support in PMTCT were 3.51 times more likely to have good adherence than those without (aOR = 3.51, 95% CI = 1.21–10.15). Moreover, there was a significantly lower odds of adherence to option B+ among those who had been on treatment between one to two years as compared to those had less than one year of treatment (aOR = 0.45; 95%CI = 0.22–0.93). Conclusion: Adherence to PMTCT option B+ antiretroviral drugs treatment among pregnant women and breastfeeding mothers was low and much lower among urban residents. Adherence was significantly predicted by rural residence, male partner support and short duration on ART. Efforts to improve adherence should focus on increasing male participation on PMTCT, tailored interventions to urban residents and those who have been on ART for a long duration. [ABSTRACT FROM AUTHOR]