Introduction A new emerging role of nitric oxide (NO) in the aetiology of osteoarthritis (OA) has been reported. Inducible NO synthase (iNOS), produced by chondrocytes, is the major source of NO in the osteoarthritic cartilage. The aim of this study is to evaluate the potential association between the -1173C/T (rs9282799), -1026 C/A (rs 2779249) and -954G/C (rs1800482) single nucleotide polymorphisms (SNPs) in the promoter of the iNOS gene (NOS2A) and the incidence of knee OA in Greek population. Methods Ninety-six patients with primary knee OA were included in the study along with 44 controls. Genotypes were identified using polymerase chain reaction (PCR) and DNA sequencing techniques. Allelic and genotypic frequencies were compared between patients and controls. Results None of the -1173C/T, -1026 C/A and -954G/C SNPs were detected in the studied population, either in patients or controls. However, another SNP was identified at the site -1056 at the promoter region, where the initial G allele was substituted by the T allele. Interestingly, the TT genotype was completely absent in controls, but was detected in six patients with a 6.2% observed frequency. The difference between patients and controls was not statistically significant (p-value = 0.18). In male OA patients, the observed frequency of the TT genotype was higher (28.6%) in comparison to the 0% of the male controls (p-value = 0.1). The frequency of the G allele was 0.82 in controls and 0.78 in OA patients (p-value = 0.53). Conclusions The present study demonstrates that the 954G/C, -1026C/A, -1056G/T and -1173C/T SNPs of the NOS2A gene are not a risk factor for primary knee OA in Greek population. Moreover, -954G/C, -1026C/A and -1173C/T are rare, if not completely absent, in the Greek population. Additional research is mandatory in order to investigate the association of these SNPs with OA in different ethnic populations.