A rare variant of chromosomal region 15q25.1, marked by rs16969968 (substitution 1354G>A in CHRNA5), was found to be associated with increased lung cancer and nicotine-dependence risk. We attempted to confirm the relationship of the polymorphism of the CHRNA5 gene and nicotine-dependence strength measured by the Fagerström test with the serum cotinine level in lung cancer and chronic obstructive pulmonary disease (COPD) patients and healthy individuals. Polymorphism of the CHRNA5 gene was analyzed using the PCR-based restriction fragment length polymorphism method in 97 lung cancer patients, 99 COPD patients, and 98 healthy individuals. The Fagerström test was used as an instrument for assessing the intensity of physical addiction. Cotinine serum level was measured using an enzyme-linked immunosorbent assay. The frequencies of AA, AG, and GG genotypes were 10.5, 47.3, and 42.2%, respectively. The polymorphism of CHRNA5 did not have a significant influence on the elevated risk of lung cancer and COPD. The percentage of smokers did not differ between groups of study participants with different genotypes. However, the presence of the GG genotype decreased the risk of nicotine addiction strength (hazard ratio=0.238; 95% confidence interval 0.066–0.857; P<0.05). Moreover, allele A was presented more frequently in participants with a high level of nicotine dependence and in participants with early addiction onset (P<0.05). Serum cotinine level was significantly correlated with the results of the Fagerström test (P<0.001). The carriers of allele A expressed significantly higher levels of cotinine when compared with the carriers of the GG genotype (P=0.05). We report for the first time the relationship between the polymorphism of the CHRNA5 gene and the strength of nicotine addiction measured by multiple factors including the Fagerström test score.