The present study investigates the potential role of Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile single-nucleotide polymorphisms (SNPs) as risk factors in the development of sarcoidosis using a novel high-throughput microtiter well-based bioluminometric genotyping assay. One hundred and nineteen Greek patients with sarcoidosis and 209 control subjects were genotyped for the two SNPs of the TLR4 gene. The genotypes observed were in Hardy-Weinberg equilibrium. The heterozygote frequency for both SNPs in sarcoidosis group and control population was 13.4% (16/119) and 10.5% (22/209), respectively. The minor genotype was found to be the same for both sarcoidosis and control groups and similar to that found in other Caucasian populations. No significant association of Asp299Gly and Thr399Ile polymorphisms with increased susceptibility to sarcoidosis was found (p = 0.61 and odds ratio = 1.183). In conclusion, genotype data for the TLR4 Asp299Gly and Thr399Ile polymorphisms in the Greek population were found to be in linkage disequilibrium, and no contribution in the pathogenesis of sarcoidosis was established. Further, in course of the present study, we demonstrated a very simple and sensitive high-throughput bioluminometric assay for genotyping Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene.