Summary The fine centromere structure in Robertsonian wheat‐rye translocation chromosomes exhibits variation among different translocation genotypes. Within extensively employed wheat‐rye 1RS.1BL translocation lines in wheat breeding, their translocated chromosomes frequently display fused centromere. Nevertheless, the mechanism governing the functionality of the fused centromere in 1RS.1BL translocated chromosomes remains to be clarified. In this study, we investigated the fine centromere structure of the 1RS.1BL translocated chromosome through a combination of cytological and genomics methods. We found that only the rye‐derived centromere exhibits functional activity, whether in breeding applications or artificially synthesized translocation chromosomes. The active rye‐derived centromere had higher proportion of young full‐length long terminal repeat retrotransposons (flLTR‐RTs) and more stable non‐B DNA structures, which may be beneficial toward transcription of centromeric repeats and CENH3 loading to maintain the activity of rye centromeres. High levels of DNA methylation and H3K9me2 were found in the inactive wheat‐derived centromeres, suggesting that it may play a crucial role in maintaining the inactive status of the wheat centromere. Our works elucidate the fine structure of 1RS.1BL translocations and the potential mechanism of centromere inactivation in the fused centromere, contributing knowledge to the application of fused centromere in wheat breeding formation of new wheat‐rye translocation lines.