Centromeres in eukaryotes mediate the accurate segregation of chromosomes during cell division. They serve as essential functional units of chromosomes and play a core role in the process of genome evolution. Centromeres are composed of satellite repeats and highly repetitive centromeric retrotransposons (CRs), which vary greatly even among closely related species. Cucumber () is a globally cultivated and economically important vegetable and the only species in the genus with seven pairs of chromosomes. Therefore, studying the centromeres of the subgenus may yield valuable insights into its genome structure and evolution. Using chromatin immunoprecipitation (ChIP) techniques, we isolated centromeric DNA from cucumber reference line 9930. Our investigation into cucumber centromeres uncovered the centromeric satellite sequence, designated as CentCs, and the prevalence of Ty1/ long terminal repeat retrotransposons. In addition, active genes were identified in the CsCENH3 nucleosome regions with low transcription levels. To the best of our knowledge, this is the first time that characterization of centromeres has been achieved in cucumber. Meanwhile, our results on the distribution of CentCs and CsCRs in the subgenus indicate that the content of centromeric repeats in the wild variants was significantly reduced compared with the cultivated cucumber. The results provide evidence for centromeric DNA amplification that occurred during the domestication process from wild to cultivated cucumber. Furthermore, these findings may offer new information for enhancing our understanding of phylogenetic relationships in the genus.