Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single-copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination-poor region is preferentially marked by a heterochromatin-typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin-typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape. Significance Statement To fully exploit the barley genome for crop improvement, it is necessary to better understand the relationship between physical and genetic distances. Here we used low-copy probes for fluorescence in situ hybridization to order contigs corresponding to a non-recombining genetic centromere of barley.