B chromosomes (Bs) are dispensable, less-transcriptionally active components of the genomes of numerous species. Little information is available on the chromatin composition of Bs and whether it differs in any way from that of the A chromosomes. Methylated isoforms of histone H3 are of particular interest because of their role in eu/heterochromatin formation. Immunofluorescence using site-specific antibodies demonstrates that the chromatin in A and both types of Bs of B. dichromosomatica differs markedly in euchromatic histone H3 methylation marks. While A chromosomes are labelled brightly, the micro B and large B chromosomes are faintly labelled with antibodies against H3K4me2/3, H3K9me3 and H3K27me2/3. The heteropycnotic, tandem-repeat enriched micro Bs were even less labelled with euchromatic histone H3 methylation marks than large Bs, most probably due to different DNA composition. No differences in immunolabelling intensity between A and B chromosomes were found as to the heterochromatic marks H3K9me1/2 and H3K27me1, indicating that Bs are not additionally labelled by heterochromatin typical histone H3 modifications. Analysis of DNA replication timing suggests that micro Bs are replicating throughout S-phase.