No software currently implements a test of linkage disequilibrium in autotetraploid species. We propose a program, LD4X that performs a Fisher's exact test between pairs of alleles at two loci. All combinations of alleles from two loci are treated in turn. If two alleles of a pair of loci have a nonrandom distribution, the markers are considered as linked. The program was tested on a set of microsatellite markers in synthetic alfalfa populations.