To investigate selenium tolerance mechanisms in Arabidopsis thaliana, genetic and physiologic studies were performed in the three Arabidopsis accessions Landsberg erecta (Ler), Columbia (Col) and Wassilewskija (Ws). Accession Ler was significantly less tolerant to selenate than Ws and Col, whereas Ws was less tolerant to selenite than the others. Analysis of selenium tolerance in F1 and F2 plants obtained from crosses between these accessions suggest that multiple genes are involved in selenate tolerance and a single major gene controls selenite tolerance in these populations. Bulked segregant analysis in two F2 populations indicated that molecular marker ciw7 on chromosome 4 is linked to selenite tolerance, and three molecular markers on chromosomes 1, 3 and 5 (nga111, ciw4 and ciw8, respectively) are linked to selenate tolerance. The ecotypic variation in selenite tolerance appeared to be correlated with root levels of non-protein thiols. Also, the shoot tissue levels of selenocysteine (SeCys) and selenocystine were correlated with tolerance to both selenate and selenite. Judging from RT-PCR results, several sulfate transporters and S assimilatory enzymes appear to be upregulated by selenate and selenite at the transcriptional level. A potential SeCys methyltransferase was expressed at lower levels in selenite-sensitive Ws than in the other two, when grown on selenite. Together, these studies show that there is substantial intraspecific variation in tolerance to selenate and selenite in Arabidopsis, and provide insights into the genetic and biochemical mechanisms underlying the observed ecotypic differences.