Atrio-ventricular conduction disease is a common feature in Mendelian rhythm disorders associated with sudden cardiac death and is characterized by prolongation of the PR interval on the surface electrocardiogram (ECG). Prolongation of the PR interval is also a strong predictor of atrial fibrillation, the most prevalent sustained cardiac arrhythmia. Despite the significant genetic component in PR duration variability, the genes regulating PR interval duration remain largely elusive. We here aimed to dissect the quantitative trait locus (QTL) for PR interval duration that we previously mapped in murine F2 progeny of a sensitized 129P2 and FVBN/J cross. To determine the underlying gene responsible for this QTL, genome-wide transcriptional profiling was carried out on myocardial tissue from 109 F2 mice. Expression QTLs (eQTLs) were mapped and the PR interval QTL was inspected for the co-incidence of eQTLs. We further determined the correlation of each of these transcripts to the PR interval. Tnni3k was the only eQTL, mapping to the PR-QTL, with an established abundant cardiac-specific expression pattern and a significant correlation to PR interval duration. Genotype inspection in various inbred mouse strains revealed the presence of at least three independent haplotypes at the Tnni3k locus. Measurement of PR interval duration and Tnni3k mRNA expression levels in six inbred lines identified a positive correlation between the level of Tnni3k mRNA and PR interval duration. Furthermore, in DBA/2J mice overexpressing hTNNI3K, and in DBA.AKR.hrtfm2 congenic mice, which harbor the AKR/J "high-Tnni3k expression" haplotype in the DBA/2J genetic background, PR interval duration was prolonged as compared to DBA/2J wild-type mice ("low-Tnni3k expression" haplotype). Our data provide the first evidence for a role of Tnni3k in controlling the electrocardiographic PR interval indicating a function of Tnni3k in atrio-ventricular conduction.