ObjectiveTo identify proportion of and factors for comprehensive knowledge on prevention of mother-to-child transmission of HIV in pregnant women attending antenatal care in Northern Ethiopia.MethodsA total of 416 pregnant women were interviewed between October 2012 and May 2013. Logistic regression analysis was used to identify factors for comprehensive knowledge on prevention of mother-to-child transmission of HIV.ResultsThe proportion of pregnant women, who have comprehensive knowledge on prevention of mother-to-child transmission of HIV, was 52%. The odds of having comprehensive knowledge on prevention of mother-to-child transmission of HIV were higher among pregnant women who were younger (16 to 24 years old) (Adjusted Odds Ratio (AOR) = 2.95; 95%CI: 1.20, 7.26), urban residents (AOR = 2.45; 95%CI: 1.39, 4.32), attending secondary education and above (AOR = 4.43; 95%CI: 2.40, 8.20), employed (AOR = 4.99;95%CI: 2.45, 10.16), have five children or more (AOR = 9.34; 95%CI:3.78, 23.07), have favored attitude towards HIV positive living (AOR = 2.53; 95%CI: 1.43, 4.44) and have perceived susceptibility to HIV (AOR = 10.72; 95%CI: 3.90, 29.39).ConclusionThe proportion of women who have comprehensive knowledge on prevention of mother-to-child transmission of HIV in this study setting was low. Measures which will escalate mother’s knowledge on prevention of mother-to-child transmission of HIV should be emphasized. Efforts to improve mother’s knowledge on prevention of mother-to-child transmission of HIV should target women who were older age (> = 35years), rural residents, unemployed, not attending formal education, primigravids, have no favored attitude towards HIV positive living and have not perceived susceptibility to HIV.