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Modern soybean [Glycine max (L.) Merr.] breeding extensively uses as crossing parents superior cultivars and breeding lines with locally adapted desirable traits, which may lead to declined genetic diversity and increased frequency of fixed common alleles. The main objective of this research was to evaluate, via amplified fragment length polymorphism (AFLP) markers, the genetic diversity and the genetic relationships among the 38 most popular cultivars and lines in the southern United States. Genetic analysis using AFLP markers with 16 primer combinations revealed an average genetic distance of 0.124 among the 38 genotypes examined. Genetic diversity declined among cultivars released during the 1950s and 1960s compared to those released before the 1950s, but increased in the 1970s and peaked in the 1980s, followed by a decline again in the 1990s and 2000s. While 34 unique bands were identified, allelic frequency analysis revealed 50.8% of the 754 loci examined to be fixed in all 38 genotypes. Future parent selection for crossing should consider using genotypes of greater genetic distance and avoid using lines with common genetic background. [ABSTRACT FROM AUTHOR]