Atrial fibrillation (AF) is the most common form of cardiac arrhythmia observed in clinical practice and a major contributor to cardiovascular morbidity and mortality. Accumulating evidence indicates a substantial genetic basis for AF. However, AF is genetically heterogeneous and the hereditary components responsible for AF remain to be identified in the majority of patients. The cardiac gap junction protein α 5 (GJA5) is specifically expressed in atrial myocytes and is associated with the coordinated electrical activation of the atria, providing a rationale to screen GJA5 as a logical candidate gene for AF. A cohort of 310 unrelated patients with lone AF and their available relatives were included in this study. A group of 200 unrelated healthy individuals matched for age, gender and race were also included as controls. The entire coding region and splice sites of the GJA5 gene were initially sequenced in 310 unrelated AF patients. The relatives of mutation carriers and 200 controls were subsequently genotyped for the presence of identified mutations. As a result, 4 novel heterozygous GJA5 mutations, p.K107R, p.L223M, p.Q236H and p.I257L, were identified in 4 of 310 unrelated AF patients, respectively, with a prevalence of ~1.29%. Genetic analysis of the carriers' families showed that in each family the missense mutation was present in all the affected family members. Absent in the 400 reference alleles, these mutations altered the amino acids highly conserved among various species, with the exception of p.I257L. In conclusion, this study expands the spectrum of GJA5 mutations associated with AF and provides novel insights into the molecular basis of AF, suggesting potential implications for the improved, gene-specific rhythm control strategies.