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Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants produced during incomplete combustion of organic matter. Humans can be exposed to them via several pathways (inhalation, digestion, dermal exposure). The aim of this study was to assess the concentration of 11 monohydroxylated metabolites of PAHs (OH-PAHs) in 660 urine samples collected from mothers and their newborns residing in two localities of the Czech Republic - Most and Ceske Budejovice - in 2016 and 2017. After enzymatic hydrolysis, the target analytes were extracted from the urine samples using liquid-liquid extraction, with extraction solvent ethyl acetate and a clean-up step using dispersive solid-phase extraction (d-SPE) with the Z-Sep sorbent. For identification and quantification, ultra-high performance liquid chromatography coupled with tandem mass spectrometry was applied. 2-OH-NAP was the compound present in all of the measured samples and it was also the compound at the highest concentration in both mothers' and newborns' urine samples (median concentration 5.15 μg/g creatinine and 3.58 μg/g creatinine). The total concentrations of OH-PAHs in urine samples collected from mothers were 2 times higher compared to their children. The most contaminated samples were collected in Most in the period October 2016-March 2017 from both mothers (12.59 μg/g creatinine) and their newborns (8.29 μg/g creatinine). The concentrations of OH-PAHs in urine samples, which were collected from both mothers and their newborns as presented in this study, are comparable with those found in our previous study between 2013 and 2014. In addition, they are slightly lower or comparable to other studies from Poland, USA, Germany, China, and Australia. The results might indicate that the population in the previously highly air-polluted mining districts carries some long-term changes (maybe existing changes in genetic information), which also affect the metabolism of PAHs. It could be related to the long-lasting effect, and thus corresponding to the shortened life expectancy., Competing Interests: Declaration of competing interest All authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)