A new high-resolution digital radiographic technique based on the deposition of ¹²⁵I- and ³H-labeled desmethylimipramine (IDMI and HDMI, respectively) was developed for the assessment of spatial and temporal myocardial flow heterogeneity at a microvascular level. The density distributions of two tracers, or relative flow distributions, were determined by subtraction digital radiography using two imaging plates of different sensitivity. The regions resolved are comparable in size to vascular regulatory units (400 × 400 µm²). This method was applied to the measurement of within-layer myocardial flow distributions in Langendorff-perfused rabbit hearts. The validity of this method was confirmed by the strong correlation between regional densities of two tracers injected simultaneously (r = 0.89 ± 0.03, n = 8). The temporal flow stability was evaluated by a 90-s continuous IDMI injection and subsequent bolus HDMI injection (n = 8). Regional densities of the two tracers were fairly correlated (r = 0.86 ± 0.03), indicating that the spatial pattern of flow distribution was stable even at a microvascular level over a 90-s period. The effect of microsphere embolization on the flow distribution was also investigated by the sequential injections of IDMI, 15-µm microspheres, and HDMI at 20-s intervals (n = 8). Microembolization increased the coefficient of variation of tracer density from 19 to 25% (P < 0.05), whereas the regional densities of two tracers were still correlated substantially, as in the case of no embolization (r = 0.84 ± 0.06). Thus the microsphere embolization enhanced flow heterogeneity with increasing flow differences between control high-flow and control low-flow regions but rather maintained the pattern of flow distribution. In conclusion, double-tracer digital radiography will be a promising method for the spatial and temporal myocardial flow analysis at microvascular levels.