The purpose of the current study was to screen newborns in Greece and to identify the responsible mutations for Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD).47.812 neonates were screened for the potential presence of MCADD in Greece, via a LC–MS/MS protocol. The “suspected” samples were subjected to genetic testing via PCR–RFLP and sequencing of the coding region of the ACADM gene. Urine samples were collected and then analyzed with a GC/MS method.The MCADD prevalence is 1 in 15,937 births. The alleles c.985A>G and c.245insT were detected in the 29.2% and 20.8% of the “suspected” cohort, respectively. A novel variant with potential pathogenicity was identified.The c.245insT allele seems to prevail in the Greek cohort of “suspected” specimens. Therefore, this variant along with the c.985A>G allele could constitute a panel for both prenatal and neonatal MCADD screening in the Greek population.