A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was determined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome.