The isolation and characterization of a library of short (< 1 kbp) DNA probes suitable for restriction fragment length polymorphism (RFLP) analysis of Trichostrongylus colubriformis are described. More than 70% of these probes contained repetitive DNA sequences, implying that repetitive DNA is widely dispersed in the T. colubriformis genome. Analysis of one laboratory and one field strain for RFLPs using non-repetitive probes showed that there is extensive genetic heterogeneity within each strain (17 of 21 probe X enzyme combinations detected multiple bands on genomic Southern blots, consistent with the existence of multiple alleles) and also considerable polymorphism between strains (18 of the 62 bands detected were strain specific). The laboratory strain was not significantly less polymorphic than the field strain, suggesting that a relatively small, restricted population size is sufficient for the maintenance of the polymorphism observed. The possible applications of these data in the epidemiology and population genetics of the parasite are discussed.