Since the density of simple sequence repeats (SSRs) may vary between different chromosomes of the same species in eukaryotic genomes, we screened SSRs of the whole genome of the yellow necked mouse, Apodemus flavicollis, in order to reveal SSR profiles specific for animals carrying B chromosomes. We found that the 2200 bp band was amplified by primer (CAG)4AC to a highly increased level in samples with B chromosomes. This quantitative difference (B-marker) between animals with (+B) and without (0B) B chromosomes was used to screen 20 populations (387 animals). The presence/absence of Bs was confirmed in 96.5% of 342 non mosaic individuals, which recommends this method for noninvasive B-presence detection. A group of 45 animals with mosaic and micro B (μB) karyotypes was considered separately and showed 55.6% of overall congruence between karyotyping and molecular screening results. Relative quantification by qPCR of two different targeted sequences from B-marker indicated that these B-specific fragments are multiplied on B chromosomes. It also confirms our assumption that different types of Bs with variable molecular composition may exist in the same individual and between individuals of this species. Our results substantiate the origin of Bs from the standard chromosomal complement. The B-marker showed 98% sequence identity with the serine/threonine protein kinase VRK1 gene, similarly to findings reported for Bs from phylogenetically highly distant mammalian species. Evolutionarily conserved protein-coding genes found in Bs, including this one in A. flavicollis, could suggest a common evolutionary pathway.