A 1.5-Mb duplication on chromosome 17p11.2-p12 (CMT1A duplication) caused by a misalignment of the CMT1A repeat sequences (CMT1A-REPs) is associated with Charcot-Marie-Tooth disease type 1A (CMT1A). A hotspot of crossover breakpoints located in a 3.2-kb region of the CMT1A-REPs accounts for three-quarters of the rearrangements in CMT1A patients. We developed a PCR-based diagnostic method to detect a recombination hotspot associated with the CMT1A duplication. Thirty-one CMT1A Chinese patients from different families and 50 healthy people over 65 years of age were studied. Twenty-seven of the 31 cases demonstrated the 3.2-kb hotspot crossover, of which there were two subgroups. The type 1 crossover breakpoint was located at the distal CMT1A-REP around the PmeI site, and accounted for 24 of the 27 cases with a 3.2-kb hotspot crossover in CMT1A duplication patients. The type 2 crossover breakpoint was located at the distal CMT1A-REP around the base 3625 region, accounting for 3 of the 27 cases. The results correlated very well with the results of Southern transfer analysis. This study has a potentially important role in the diagnosis of CMT1A disease.