The present study aimed to determine frequencies of mutations in the phenylalanine hydroxylase gene (PAH) in unrelated children (n = 71) diagnosed with phenylketonuria, who presented to Morozovskaya Children’s City Clinical hospital (Moscow) over the period from 2015 to 2016. The patients were tested for the most common PAH mutations using the original real- time PCR-based technique for the identification of nucleotide variants; additionally, next generation sequencing (NGS) was performed on the unidentified genotypes. The original PCR-based technique allowed us to effectively identify 83 % of the pathogenic allelic variants in the sample. Using the combination approach (real-time PCR + NGS), we found mutations in both alleles of PAH in 66 of total 71 patients. Altogether, 26 pathogenic PAH mutations were identified, the most common being p.R408W (47.9 %) and p.R261Q (9.9 %). Frequencies of mutations common for the Russian population, such as IVS10nt546, IVS12+1G>A, p.R158Q, p.Y414C, and IVS4+5G>T, ranged from 4.2 to 2.8 %. Half of the identified variants accounted for the total frequency of < 10 %. Sequencing of PAH revealed a few functional mutations previously unreported for Moscow region residents, including p.D222Terfs, p.R111Ter, p.F161S, p.G188D, p.R270K, p.L311P, p.F55L, p.F55Leufs, IVS1+5G>T, and IVS8-7A>G. It could be reasonable to include mutations p.D222Terfs and p.R111Ter (carrier frequency of 2.1 %) in PCR testing panels. The data obtained in our study can also be used in the development of genetic tests for phenylketonuria.