Background: Premature birth remains a serious public health problem in developing countries including Ethiopia. Ethiopia is one of the country with highest preterm birth rate in Africa. However, there is limited evidences on the prevalence and associated factors of premature birth. Therefore, this study was aimed to assess the prevalence and associated factors of premature birth among newborns delivered in Amhara region referral hospitals, Northern Ethiopia.Methods: A hospital-based cross-sectional study was undertaken from February 23rd to April 23rd, 2020, in the Amhara region. A total of 482 mother-newborn pairs were included in this study. The data were collected by interviewing the mothers and reviewing their charts using a structured and pretested questionnaire. The outcome variable was preterm birth. Data were entered using Epi-data version 4.6 and analyzed using STATA software (version 14). Bivariable and multivariable logistic regression models were done for the factors associated with premature birth. Results: In this study, the prevalence of premature birth was 11.41%(95% CI: 8.9, 14.6%). In multivariable logistig regression model; maternal age < 20 years (Adjusted odds ratio (AOR) = 7.8: 95% CI 2.3 – 26 ), preeclampsia (AOR = 5: 95% CI 2.3 – 11 ), premature rupture of membrane (AOR=3.9: 95%CI 1.6- 9.0), chronic medical illness (AOR=4.6:95% CI2.1-10), and history of stillbirth (AOR = 2.7: 95% CI 1.1-7.3) were significantly associated with preterm birth.Conclusion: The finding of this study showed that the burden of premature birth is a public health concern among newborns delivered in Amhara region referral hospitals. Maternal age less than 20 years, preeclampsia, premature rupture of membrane, chronic medical illness, and history of stillbirth were factors associated with preterm birth. Therefore, efforts have to be made to reduce the burden of prematurity, and for early detection and management of preeclampsia. Premature rupture of membrane, and chronic medical illness. Obstetric care providers should give due attention to women with an age of less than 20 years and a history of stillbirth.