The habitat fragmentation that characterizes agricultural systems made up of an array of sympatric crop and weed biotopes might be a major determinant of the population structure of plant-feeding arthropods. Spider mites are interesting in this regard as several species are highly polyphagous, possibly generating homogenization by plant shifting, but also have the potential of forming host plant races which promotes specialization. This study analyses genetic diversity and structure in natural populations of a spider mite pest, Tetranychus turkestani, collected on both crops and weeds. Five microsatellite markers were used to genotype 283 individuals collected from 15 samples in four locations in southern France. The markers revealed considerable genetic variation, with an average heterozygosity of 0.68. Pairwise ΦST estimates calculated between localities showed differentiation in all comparisons. Although geographical distance appears to be a factor that influences T. turkestani population genetic structure at a regional scale, there was no clear evidence for differentiation between mites living on different host plants. A hierarchical analysis of the distribution of the genetic diversity within and between habitats showed that more than 97% of the observed genetic variation accounted for the differentiation between mites collected on the same host plant in a given locality. Significant heterozygote deficiency was found in 11 out of the 15 samples studied. Considering the biology of the mite, a Wahlund effect and inbreeding might explain such an excess of homozygosity. The data support the view that the plants in a given locality are colonized by mites that originate from diverse sources. They also support previous data suggesting that the demographic structure is made up of small demes of inbred individuals. The agricultural-level implications of the data are discussed, notably the fact that mites may well be capable of moving between host plants, making weeds surrounding the crop fields potential reservoirs for the pest. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 69–78.