Rationale: Ventilation-perfusion scintigraphy is highly sensitive for pulmonary embolism, but its clinical usefulness is limited by its non-diagnostic rate. Objective analysis of Single Photon Emission Computed Tomography (SPECT) three-dimensional scintigraphy may improve its diagnostic performance compared to subjective interpretation. Objectives: To determine the diagnostic accuracy of objective SPECT analysis in pulmonary embolism. Methods: We determined the ventilation/perfusion (V/Q ) relationship using SPECT scintigraphy in a retrospective cohort of 73 patients. Measures of V/Q heterogeneity (LogSD_Q, LogSD_V, LogSD_VQR) including a novel parameter, the weighted median V/Q value, were calculated. Using receiver operator characteristic (ROC) analysis, each parameter's diagnostic accuracy was determined. The weighted median V/Q value was then assessed prospectively in a second cohort of 50 patients. Main Results: In cohort one, all parameters of V/Q heterogeneity were higher in patients with pulmonary embolism (p<0.002). The weighted median V/Q had the highest area under the ROC curve (0.93, 95%CI 0.87 - 0.98). When applied to the prospective cohort, the area under the ROC curve was 0.87 (95%CI 0.75 - 0.99), with diagnostic cut-off values having negative and positive predictive values of 96% and 83% respectively. In the retrospective and prospective cohorts, 82% and 73% of initially reported intermediate or low probability scans had diagnostic weighted median V/Q values, with 90% and 100% accuracy respectively. Conclusions: Objective analysis of SPECT scintigraphy has a high diagnostic accuracy in patients with suspected pulmonary embolism. Objective analysis has the potential to reduce the number of non-diagnostic scan results, and may be useful for quantifying V/Q mismatch in other pulmonary disorders.