Background: Although Ethiopia is implementing an option B+ program for the last seven years, lost to follow-up among HIV positive women remains the major problem for ART treatment. The study was conducted on lost to follow-up among human immunodeficiency virus infected pregnant and breastfeeding women after Option B+ modality since there was a scarcity of literatures in Northwest Ethiopia. The result is also expected to strengthen the effort to eliminate mother-to-child transmission of HIV to 5%. Methods: A retrospective follow-up study was conducted among 403 pregnant and breastfeeding women between June 2013 and December 2019 at the University of Gondar comprehensive specialized Hospital. Cox proportional hazards regression model was used for identifying predictors of lost-to-follow-up. Results were reported as hazard ratios with 95% confidence interval at a significance level of p=0.05. Results: The overall incidence rate of loss to follow-up was 9.4 per 1000 person-months of observation (95% confidence interval (CI): 7. 4‒11. 9). According to the multivariable Cox regression, rural residency (adjusted hazard ratio(AHR): 2.30; 95% CI:1.08‒4.88), being Muslim religion follower (AHR: 2.44; 95% CI:1.23‒4.81), having no baseline viral load measurement (AHR: 4.21; 95% CI:2.23–7.96, being on ART before enrolment (AHR: 0.30; 95% CI:0.15–0.62), having drug side effects (AHR: 1.82; 95% CI:1.01–3.33), same day ART initiation (AHR: 3.23; 95% CI:1.53–6.84) and having sub-optimal adherence level (AHR: 3.96; 95% CI:2.18–7.19) were significant predictors of lost to follow up.Conclusion: The incidence of lost to follow-up is lower as compared to evidence from most African countries but slightly higher than the WHO target. It is better to strength and expand viral load measurement for all women and giving attention to those women who are residing in a rural area and having fair/poor adherence level.