Trifolium medium L. is a wild polyploid relative of the agriculturally important red clover that possesses traits promising for breeding purposes. To date, T. medium also remains the only clover species with which agriculturally important red clover has successfully been hybridized. Even though allopolyploid origin has previously been suggested, little has in fact been known about the T. medium karyotype and its origin. We researched T. medium and related karyotypes using comparative cytogenomic methods, such as fluorescent in situ hybridization (FISH) and RepeatExplorer cluster analysis. The results indicate an exceptional karyotype diversity regarding numbers and mutual positions of 5S and 26S rDNA loci and centromeric repeats in populations of T. medium ecotypes and varieties. The observed variability among T. medium ecotypes and varieties suggests current karyotype instability that can be attributed to ever-ongoing battle between satellite DNA together with genomic changes and rearrangements enhanced by post-hybridization events. Comparative cytogenomic analyses of a T. medium hexaploid variety and diploid relatives revealed stable karyotypes with a possible case of chromosomal rearrangement. Moreover, the results provided evidence of T. medium having autopolyploid origin.