BACKGROUND: Despite an estimated 59,000 incident pediatric HIV infections in 2012 in Nigeria, rates of early infant diagnosis (EID) of HIV service uptake remain low. We evaluated maternal factors independently associated with EID uptake in rural North Central Nigeria. METHODS: We performed a cohort study using HIV/AIDS program data of HIV-infected pregnant women enrolled into HIV care/treatment on or before December 31, 2012 (n = 712). We modeled the probability of initiation of EID using multivariable logistic regression. RESULTS: Three hundred fifty-seven HIV-infected pregnant women enrolled their infants in EID across the 4 study sites. Women who enrolled their infants in EID vs. those who did not were similar across age, occupation, referral source, and select laboratory variables. Clinic of enrollment and date of enrollment were strong predictors for EID entry (P < 0.001). Women enrolled more recently were less likely to have their infants undergo EID than those enrolled at the beginning of the project (January 2011 vs. January 2010, adjusted odds ratio = 0.35, 95% confidence interval: 0.22 to 0.56; January 2012 vs. January 2010, adjusted odds ratio = 0.30, 95% confidence interval: 0.14 to 0.61). Women who received care in the more urban setting of Umaru Yar Adua Hospital were more likely to have their infants enrolled in EID than those who received care in the other 3 clinics. CONCLUSIONS: HIV-infected women in our prevention of mother-to-child HIV transmission program were more likely to bring in their infants for EID if they were enrolled in a more urbanized clinic location, and if they presented during an earlier phase of the program. The need for more intensive family engagement and program quality improvement is apparent, especially in rural settings.