Crossovers (COs) generated by homologous recombination ensure the proper segregation of chromosomes during meiosis. COs exhibit interference, which leads to widely spaced COs along chromosomes. Strong positive CO interference has been found in humans. However, little is known about the extent of human CO interference. In this study, variations in CO interference over the entire human genome and among individuals were analyzed by immunofluorescence combined with fluorescence in situ hybridization of testicular biopsies from 10 control men. These methods allow for direct identification of the frequency and location of COs in specific chromosomes of pachytene cells. The strength of CO interference was estimated by fitting the frequency distribution of inter-CO distances to the gamma model. Positive interference among CO on chromosomes was observed in these men, and the strength of inter-arm interference was significantly stronger than that for intra-arm CO. In addition, interference was observed to act across the centromere. Significant inter-individual and inter-chromosomal variations in the levels of interference were found, with smaller chromosomes exhibiting stronger interference. Discontinuous chromosome regions (gaps) and unsynapsed chromosome regions (splits) in chromosome 9 had both cis and trans effects on CO interference levels. This is the first report that the interference level varies significantly across the whole genome and that, at least in the human male, anomalies in chromosome synapsis play an important role in altering CO interference levels.