Cotton (Gossypium hirsutum L.) is crucial for the textile industry worldwide. Among the diseases attacking cotton, Verticillium wilt caused by Verticillium dahliae Kleb. is the most significant. Isolates of V. dahliae can be classified into defoliating and non-defoliating pathotypes. Thirty-two isolates of the non-defoliating pathotype and one isolate of a virulent, defoliating pathotype were analysed by the amplified fragment length polymorphism (AFLP) method. Three hundred and forty AFLP fragments were obtained with nine primer combinations. The number of total bands per primer pair ranged from 16 to 81, with an average of 37.7. The average polymorphism information content (PIC) value for the AFLP products was 0.50. Using the genotypic data, genetic distance analysis was performed. The maximum variation was found between isolates (Vd11) Nazilli and (Vd16) Soke, at a value of 0.79 and the minimum variation was found between isolates (Vd20) Aydin and (Vd14) Soke, at 0.24. The unweighted paired group method with arithmetic averages cluster analysis (UPGMA) was used to discriminate the V. dahliae isolates into five subgroups. Defoliating pathotypes (Vd33) from Soke province formed a single subgroup. As a result, it was found that there was significant variation among Verticillium isolates. AFLP analysis is an efficient and effective marker technology for determining genetic relationships among Verticillium isolates.