In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realising the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon our ability to recognise real changes in recombination patterns. We have isolated the barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, and raised a specific antibody. Immunolocalisation of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridisation (FISH) tagging of specific barley chromosomes, has enabled us to access the physical recombination landscape of the barley cultivars Morex and Bowman. We have observed consistent distal localisation of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H. We have quantified a difference in total numbers of HvMLH3 foci between these two cultivars, which we interpret as representing genotypic variation in Class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. We also show that interference of HvMLH3 foci is relatively weak compared to other plant species.