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The developmental timeline of the human heart remains elusive. The heart takes on its characteristic four chambered appearance by ~56 days gestational age (DGA). However, owing to the complexities (both technical and logistical) of exploring development in utero, we understand little of how the ventricular walls develop. To address this, we employed diffusion tensor magnetic resonance imaging to explore the architecture and tissue organization of the developing heart aged 95–143 DGA. We show that fractional anisotropy increases (from ~0.1 to ~0.5), diffusion coefficients decrease (from ~1 × 10−3mm2/sec to ~0.4 × 10−3mm2/sec), and fiber paths, extracted by tractography, increase linearly with gestation, indicative of the increasing organization of the ventricular myocytes. By 143 DGA, the developing heart has the classical helical organization observed in mature mammalian tissue. This was accompanied by an increase in connexin 43 and connexin 40 expression levels, suggesting their role in the development of the ventricular conduction system and that electrical propagation across the heart is facilitated in later gestation. Our findings highlight a key developmental window for the structural organization of the fetal heart.