Supernumerary chromosomes (B chromosomes) have been an intriguing subject of study. Our understanding of the molecular differentiation of B chromosomes from an interpopulation perspective remains limited, with most analyses involving chromosome banding and mapping of a few sequences. To gain insights into the molecular composition, origin, and evolution of B chromosomes, we conducted cytogenetic and next-generation sequencing analysis of the repeatome in the grasshopper Abracris flavolineata across various populations. Our results unveiled the presence of B chromosomes in two newly investigated populations and described new satellite DNA sequences. While we observed some degree of genetic connection among A. flavolineata populations, our comparative analysis of genomes with and without B chromosomes provided evidence of two new B chromosome variants. These variants exhibited distinct compositions of various repeat classes, including transposable elements and satellite DNAs. Based on shared repeats, their chromosomal location, and the C-positive heterochromatin content on the B chromosome, these variants likely share a common origin but have undergone distinct molecular differentiation processes, resulting in varying degrees of heterochromatinization. Our data serves as a detailed example of the dynamic and differentiated nature of B chromosome molecular content at the interpopulation level, even when they share a common origin.