BACKGROUND: Adherence to antiretroviral therapy is very essential to achieve a great outcome of drugs via suppressing viral load, preventing multidrug resistance, and reducing mother to a child transmission rate of the Human Immune Virus. OBJECTIVE: This study aimed to assess the level of adherence to option B plus PMTCT and associated factors among HIV Positive pregnant and lactating women in public health facilities of Hawassa city, Southern Ethiopia, 2020 G.C. METHODS: Institution-based cross-sectional study was done on 254 HIV-positive pregnant and lactating women attending the prevention of mother-to-child transmission (PMTCT) follow-up. Participants were selected by simple random sampling. Data collected through a structured interviewer-administered questionnaire were cleaned and entered into Epi-data 3.1 and exported to SPSS 20 for statistical analysis. Descriptive analysis was done. Bivariable and multivariable logistic regressions were done to measure the strength of association between independent and dependent variables using the odds ratio and 95% of confidence interval. A p-value <0.05 was taken as statistically significant. RESULT: The overall adherence level to option B+ was 224 (88.2%). Respondents in age group of ≤ 25 [AOR = 0.12, 95% CI (0.03, 0.42)], with no formal education [AOR = 0.12, 95% CI (0.03, 0.51)], experienced drug side effects [AOR = 0.11, 95% CI (0.04, 0.32)], have good knowledge of PMTCT [AOR = 3.6, 95% CI (1.16, 11.3)], and get support from partner/family [AOR = 4.5, 95% CI (1.62, 12.4)] were identified associated factors with adherence level. CONCLUSION: The level of adherence to option B plus PMTCT was 88.2% which is suboptimal. Ages, educational level, knowledge on PMTCT, getting support from partner/family, and drug side effect were significantly associated with adherence. Therefore, educating and counseling on the service of PMTCT to improve their knowledge and encouraging partner/family involvement in care are mandatory to achieve the standard adherence level.