Atrioventricular reentry tachycardia (AVRT) requiring an accessory atrioventricular pathway (AP) is the most common type of arrhythmia in the perinatal period. The etiology of these arrhythmias is not fully understood as well as their capability to dissipate spontaneously in the first year of life. Temporary presence of APs during annulus fibrosus development might cause this specific type of arrhythmias. To study the presence of APs, electrophysiological recordings of ventricular activation patterns and immunohistochemical analyses with antibodies specifically against atrial myosin light chain 2 (MLC-2a), Periostin, Nkx2.5, and Connexin-43 were performed in embryonic mouse hearts ranging from 11.5 to 18.5 days post-conception (dpc). The electrophysiological recordings revealed the presence of functional APs in early (13.5-15.5 dpc) and late (16.5-18.5 dpc) postseptated stages of mouse heart development. These APs stained positive for MLC-2a and Nkx2.5 and negative for Periostin and Connexin-43. Longitudinal analyses showed that APs gradually decreased in number (p = 0.003) and size (p = 0.035) at subsequent developmental stages (13.5-18.5 dpc). Expression of periostin was observed in the developing annulus fibrosus, adjacent to APs and other locations where formation of fibrous tissue is essential. We conclude that functional APs are present during normal mouse heart development. These APs can serve as transient substrate for AVRTs in the perinatal period of development.