The complications introduced by the autotetraploid, outcrossing nature of alfalfa (Medicago sativa L.) as related to detecting associations of marker loci and traits of interest are discussed, and a new method of detecting marker-trait associations is suggested. This method utilizes plant populations that are likely to have been produced through the plant breeding process: populations selected for one trait, and the base, unselected population. Marker allele frequency shifts between the populations are indicative of genomic regions involved in trait expression, and may indicate alleles that have reached the triplex or homozygous state and do not segregate in S1 or F1 populations. However, because many, perhaps hundreds, of sequential frequency comparisons are needed to detect fragments in significantly different proportions in the two populations, the type I error rate is very high. A resampling-based analysis method is proposed to address the concern of the type I error rate, and identify marker alleles associated with this trait of interest. The utility of marker-trait associations thus defined for identifying individual plants from heterogeneous populations was investigated through model-building and conditional probability studies. Factors investigated that influenced the utility of the marker associations and (in the base population) the frequencies of the trait and marker, and the frequencies of the markers in plants exhibiting the trait and in the plants not exhibiting the trait. The frequency of occurrence of a marker in undesirable plants profoundly influenced the efficiency with which the marker could be used to select desirable plants, however, under some circumstances, markers or combinations of markers can be highly efficient for selecting rare, desirable plants from a heterogeneous base population.