Assessing the genetic variability of the tick Ixodes ricinus—an important vector of pathogens in Europe—is an essential step for setting up antitick control methods. Here, we report the first identification of a set of SNPs isolated from the genome of I. ricinus, by applying a reduction in genomic complexity, pyrosequencing and new bioinformatics tools. Almost 1.4 million of reads (average length: 528 nt) were generated with a full Roche 454 GS FLX run on two reduced representation libraries of I. ricinus. A newly developed bioinformatics tool (DiscoSnp), which isolates SNPs without requiring any reference genome, was used to obtain 321 088 putative SNPs. Stringent selection criteria were applied in a bioinformatics pipeline to select 1768 SNPs for the development of specific primers. Among 384 randomly SNPs tested by Fluidigm genotyping technology on 464 individuals ticks, 368 SNPs loci (96%) exhibited the presence of the two expected alleles. Hardy–Weinberg equilibrium tests conducted on six natural populations of ticks have shown that from 26 to 46 of the 384 loci exhibited significant heterozygote deficiency.