Fluorescence in situ hybridization (FISH) has been an efficient way for integrating physical and genetic maps of various small genomes like rice, sorghum and Brachypodium; whereas in the large genomes like barley, the repetitive nature of the genome complicates the generation and detection of single-copy FISH probes. Here, we used exemplarily physical map contigs of a defined interval of the long arm of barley chromosome 2H to evaluate the potential of FISH-based mapping as a supportive means for genetic anchoring of the physical map and to resolve the linear order of contigs along the respective chromosome. Repeat-free FISH probes corresponding to 8 previously anchored BAC contigs were specifically allocated to chromosome 2H. This represented an almost 90% success rate in single-copy FISH probe development. FISH mapping of contigs located in the subtelomeric region revealed an over-performance of genetic mapping over FISH for physical map anchoring. (C) 2014 S. Karger AG, Basel