Late-onset multiple carboxylase deficiency, also known as biotinidase (BTD) deficiency, is an autosomal recessively inherited disorder of biotin metabolism. Its early diagnosis and treatment seems that it can even fully prevent its various clinical manifestations. Mutations in the BTD gene scattered throughout its coding region have been detected in patients ascertained either through newborn screening or clinically. From March 2010 up to June 2011, 18 954 Greek neonates were subjected to biochemical determination of BTD activity through a semiquantitative fluoroimmunoassay. Subsequently, the first cohort of our 'suspected' samples was further tested for the presence of aberrations associated either with partial or profound BTD deficiency through sequencing of the coding region of the BTD gene, including splice-site junctions. On the basis of the molecular data derived from the study of our first cohort of 'suspected' samples, a panel of four mutations, most frequently encountered in the Greek population, was created, and a rapid, reliable and cost-effective real-time-based genotyping assay for the detection of these mutations was developed. This is the first report about the BTD mutational spectrum in Greece, and it could be a beneficial utility in the differential clinical diagnosis of BTD deficiency.