In this study, we analyzed eight samples of Hippophae rhamnoides L. collected in Kazakhstan, along with twenty-five samples of Hippophae species and three outgroup species from NCBI GenBank, using ITS sequences. The alignment of the ITS sequences, which was 639 bp in length with outgroup samples and 642 bp without them, revealed polymorphism, with 31 positions (4.85%) being polymorphic among the ingroup samples. The highest number of polymorphic sites was found in ITS1 (13 sites), followed by ITS2 (10 sites) and the 5.8S rRNA region (8 sites). The maximum likelihood (ML) phylogenetic tree delineated four distinct clades within the Hippophae species. The H. rhamnoides samples from northeastern Kazakhstan clustered with H. rhamnoides ssp. mongolica from GenBank suggests a close genetic relationship. Introduced samples formed separate subclades and clustered with various subspecies from GenBank. Notably, hybrid peaks were observed in the ITS sequences of introduced plants, which were not present in samples from natural populations. This study underscores the utility of ITS sequences in identifying plants from natural and introduced populations of H. rhamnoides and highlights the marker's importance in plant genetic research and biodiversity conservation.