Genetic variation in Lactuca serriola, the closest wild relative of cultivated lettuce, was studied across Europe from the Czech Republic to the United Kingdom, using three molecular marker systems, simple sequence repeat (SSR, microsatellites), AFLP and nucleotide-binding site (NBS) profiling. The ‘functional’ marker system NBS profiling, targeting disease resistance genes of the NBS/LRR family, did not show marked differences in genetic diversity parameters to the other systems. The autogamy of the species resulted in low observed heterozygosity and high population differentiation. Intra-population variation ranged from complete homogeneity to nearly complete heterogeneity. The highest genetic diversity was found in central Europe. The SSR results were compared to SSR variation screened earlier in the lettuce collection of the Centre for Genetic Resources, the Netherlands (CGN). In the UK, practically only a single SSR genotype was found. This genotype together with a few other common SSR genotypes comprised a large part of the plants sampled on the continent. Among the ten most frequent SSR genotypes observed, eight were already present in the CGN collection. Overall, the CGN collection appears to already have a fair representation of genetic variation from NW Europe. The results are discussed in relation to sampling strategies for improving genebank collections of crop wild relatives.