The European hornbeam (Carpinus betulus L.) is a medium-sized deciduous tree that spreads northeast of the middle of Lithuania. Carpinus betulus L. is a native tree in Poland, and its branch is migrated by two Pleistocene refugia. We hypothesised that its branches had spread to Lithuania. In this study, we selected 10 populations of hornbeam that were chosen from their distribution location. We sequenced the chloroplast intergenic spacer psbA-trnH of 70 individuals. We found 24 bp deletion in chloroplast DNA (cpDNA) individuals of two populations in the southeastern part of Lithuania. In the seven forest populations, we examined the morphological variability of hornbeam seed involucres and nuts variations of 30 morphometric characteristics. Initial genetic population studies were conducted over a wider area; when differences were detected, morphological studies were conducted in the contact zone. Morphometric differences between the study populations were significant. The existence of two haplotypes of cpDNA supports the hypothesis of two migration refugia in C. betulus populations. This study contributes to significant novel knowledge about the morphological and cpDNA variability of European hornbeam populations in Lithuania and Europe.