--- - |2+ Key points Prior studies have shown variation in the functional properties of cardiomyocytes isolated from different regions of the left ventricular myocardium. We found that these region‐dependent variations vanish below a tissue volume of ∼7 mm3 in the adult rat myocardium, revealing a fixed level of intrinsic relaxation rate heterogeneity that is independent of tissue volume. Within these microscopically varying cell populations, fast‐relaxing cells were shown to have elevated phosphorylated troponin I compared to slow‐relaxing cells. Relaxation rate was also correlated with cardiomyocyte length, in that slow‐relaxing cells were longer than fast‐relaxing cells. These results show a new relationship between cardiomyocyte morphology and myofilament relaxation, and suggest that functional diversity among individual myocytes at the microscale may contribute to bulk relaxation of the myocardium. Abstract The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. We designed experiments to quantify the variance in contractile properties among cells within the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free walls of adult Sprague–Dawley rats and then treated with collagenase to isolate individual myocytes. Cardiomyocytes were characterized by measuring sarcomere length changes and calcium transients during electrical pacing. Variance of the time from peak sarcomere shortening to 50% re‐lengthening (RT50) was assessed in each cell population. Isolating cells from progressively shorter strips allowed an estimate of the myocardial volume below which regional variation vanished and only microscale heterogeneity remained (∼7 mm3). The SD of RT50 within this myocardial volume was 28% of the mean. In a series of follow‐up experiments, RT50 was shown to correlate significantly with resting myocyte length, suggesting a connection between cell morphology and intrinsic relaxation behaviour. To explore the mechanistic basis of varying RT50, a novel single‐cell aspirator was employed to collect small batches of cardiomyocytes grouped according to their relaxation rates (fast or slow). Western blot analysis of the two groups revealed significantly elevated troponin I phosphorylation in fast‐relaxing cells. Our observations suggest that cell‐to‐cell heterogeneity of active contractile properties is substantial, with implications for how we understand myocardial relaxation and design drug therapies intended to alter relaxation rate. - 'The Journal of Physiology, EarlyView. '