OBJECTIVE:: To describe a novel technique for real-time, ultrasound-guided femoral vein catheterization in neonates with cardiac disease, and to compare it to a contemporaneous cohort of neonates undergoing femoral vein central venous line placement via landmark technique. DESIGN:: Retrospective cohort study of data extracted from a quality improvement database. SETTING:: Pediatric cardiac intensive care unit and cardiovascular operating room in pediatric tertiary hospital. PATIENTS:: One hundred fifteen neonates (mean weight, 3.07 ± 0.41 kg) with cardiac disease who underwent femoral central venous line attempts from January 2009 to September 2011. MEASUREMENTS AND MAIN RESULTS:: Study populations were similar in age, weight, and Risk Adjustment for Congenital Heart Surgery-1 category, but differed in intubation status (32% vs. 100%, ultrasound vs. landmark, p < .0001). Central venous line success rate was superior in the ultrasound group: 72 of the 76 (94.7%) vs. 31 of the 39 (79.5%), p = .02. Ultrasound group also had a superior first (75% vs. 30.8 %) and second attempt success rate (90.8% vs. 51.3%), p value for both < .0001. Inadvertent arterial puncture occurred less frequently in the ultrasound group: four of the 76 (5.3%) vs. nine of the 39 (23.1%), p = .01. There was a trend toward more venous thrombosis in the landmark group, 16 of the 39 (41%) vs. 18 of the 76 (23.7%), p = .08. Among all 115 subjects, there was a very strong association between greater than two central venous line attempts and the odds of being diagnosed with a deep venous thrombosis (odds ratio, 9.3; 95% confidence interval 3.5-24.8) and the odds of suffering an inadvertent femoral arterial puncture during the central venous line event (odds ratio, 8.8; 95% confidence interval 10.6-730). CONCLUSIONS:: This novel long-axis real-time ultrasound technique facilitates placement of femoral vein central venous line in critically ill neonates with cardiac disease at a higher rate of success with fewer attempts and lower occurrence of complications when compared with the landmark technique. © 2012 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.