Tenaw Gualu, Abebe Dilie, Dessalegn Haile, Abebe Abate Department of Nursing, College of Health Sciences, Debre Markos University, Debre Markos, Ethiopia Background: Prelacteal feeding (PLF) is giving liquids or food other than breast milk prior to the establishment of regular breast-feeding (colostrum). The practice of PLF is common in Ethiopia. PLF deprives the child of the valuable nutrients and the protection of colostrum and exposes the newborn to the risk of infection. There are limited studies conducted so far on this topic, and even then, findings are inconsistent. Objective: The objective of this study was to assess determinants of PLF practice and associated factors among postpartum mothers in Debre Markos town, Amhara Regional State, Ethiopia, 2016. Methods: A community-based cross-sectional study was conducted among 262 mothers (postpartum) with infants. Census data was used to include all the postpartum mothers and infant pairs. Structured interviewer-administered questionnaires were used to collect data. The data were cleaned, coded, and entered in Epi data version 3.1 and transferred to SPSS version 20.0 for analysis. Frequency and percentage were used to summarize the sociodemographic characteristics. Variables with a P-value of <0.05 in multivariate analysis were declared statistically significant at a 95% confidence interval.Result: Approximately 50 (19.1%) of the mothers had given one prelacteal feed before initiating colostrum. Cow milk, butter, clean water, sugar, honey, salt, and tea were the most common prelacteal feeds used. Inability to read and write 3.5 (1.14–10.75), giving birth to a male 2.8 (1.23–6.37), home delivery 4.4 (1.78–10.85), breast-feeding initiation after 24 hours 6.4 (2.38–17.18), and previous experience with PLF 3.7 (1.48–9.22) were factors positively associated with PLF.Conclusion and recommendation: It was observed that the prevalence of PLF was relatively high in the study area (19.1%). Education status of the mother, infant’s sex, timing of breast-feeding initiation, site of delivery, and previous experience with PLF were factors associated with PLF. Education should be provided to promote institutional delivery and increase awareness about the importance of exclusive breast-feeding. Keywords: prelacteal feeding, practice, factors, Debre Markos, Ethiopia