: Assessing the genetic variability of the tick Ixodes ricinus - an important vector of pathogens in Europe - is an essential step for setting up anti-tick control methods. Here we report the first identification of a set of SNPs isolated from the genome of I. ricinus, by applying a reduction of 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 (Read2Snps), 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 1,768 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 6 natural populations of ticks have shown that from 26 to 46 of the 384 loci exhibited significant heterozygote deficiency. This article is protected by copyright. All rights reserved.