Amplified fragment length polymorphism (AFLP) was used to examine genetic variation in two forms of Melampsora rust on Salix viminalis, the ‘stem-infecting form’ (SIF) and the f. sp. larici-epitea typica (LET) of M. epitea. A simple two-tube method was used to obtain genomic DNA suitable for AFLP. Eleven SIF and 26 LET isolates from the UK were tested using two primer combinations. Of the 215 AFLP markers scored, 93% were polymorphic. AFLP profiles were distinct between SIF and LET (Nei & Li's similarity coefficients between SIF and LET isolates = 22–35%). Within SIF, AFLP patterns were very similar (similarity > 98–9%), indicating that SIF is an asexual population and may have a clonal lineage. Within LET, similarities were > 69%. LET isolates collected from a site at Long Ashton in 1991–1993 were closely related and, therefore, may have come from the same local source. Similarity data from AFLP were in good agreement (Spearman's rank correlation = 0.85) with that of RAPD when both SIF and LET isolates were included, but the correlation was less obvious (= 0.49) when only LET isolates were included. Identical banding patterns were obtained when AFLP was performed using either a Perkin–Elmer 480 or a Perkin–Elmer 9700 Thermocyclers, each programmed with a different PCR profile.