Abstract Background The global breastfeeding recommendation states that all infants should be put to the breast within one hour of birth, which is defined as timely initiation or early initiation of breastfeeding. Early initiation of breastfeeding is associated with reduced risk in infant illness and death. Understanding the determinants of delay in initiation of breastfeeding might spur health staff and policy makers to foster timely breastfeeding. We assessed the prevalence and determinants of delay in initiation of breastfeeding among mothers in Juba Teaching Hospital. Methods The present study enrolled 806 mother-infant pairs within 24 hrs of birth in Juba Teaching Hospital in 2017. The mothers were interviewed about the time of initiation of breastfeeding, sociodemographic and birth characteristics. The independent variables associated with delay in initiation of breastfeeding were identified using multivariable logistic regression analysis. Results In the current study, 52% (418/806) of the mothers initiated breastfeeding later than one hour after birth. Birth by Caesarean section (Adjusted Odds Ratio [AOR] 41; 95% Confidence Interval [CI] 12.21, 138), discarding of colostrum (AOR 9.89; 95% CI 4.14, 23.62), unmarried mothers (AOR 3.76; 95% CI 1.53, 9.24), exposure to infant formula advertisement (AOR 1.82; 95% CI 1.09, 3.02) and no house ownership (AOR 1.52; 95% CI 1.11, 2.09) were independent factors associated with delay in initiation of breastfeeding. Conclusion We found that more than half of the mothers delayed the initiation of breastfeeding. Therefore, we recommend training on best breastfeeding practices and counselling skills for health staff in Juba Teaching Hospital. Policy dialogue, with the relevant ministries and departments on the promotion and protection of early initiation of breastfeeding is crucial.