Vitamin B12 deficiency causes decreased Methionine Synthase and L-Methylmalonyl-CoA Mutase activity and results in accumulation of Homocysteine, Methylmalonic acid and Propionylcarnitine. Propionylcarnitine is included in tandem mass spectrometry-based newborn screening programs for detection of certain inborn errors of metabolism. We report two asymptomatic newborns with Vitamin B12 deficiency due to maternal deficiencies. One was detected incidentally at 3 weeks of age; the second on supplemental newborn screening based on elevated Propionylcarnitine at 2 days of age. This illustrates the potential for false negative results for Vitamin B12 deficiency screening by acylcarnitine profiling in newborn screening. Homocysteine and Methylmalonic acid may be better markers of Vitamin B12 deficiency. In conclusion, we suggest measuring Methylmalonic acid, Propionylcarnitine and Homocysteine levels in blood spots in expanded newborn screening in order to detect asymptomatic newborns with Vitamin B12 deficiency. Further studies are needed to establish the sensitivity of these three markers in screening for Vitamin B12 deficiency.