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Objective We evaluated the impact of radiotracer injection latency and post-injection seizure duration on subtraction ictal SPECT co-registered to MRI (SISCOM) test performance in identifying the epileptogenic zone (EZ) in children with drug-resistant epilepsy who had undergone a resective epilepsy surgery. Methods SISCOM concordance with the EZ was retrospectively reviewed to evaluate its performance in 113 children. The impact of radiotracer injection latency and post-injection seizure duration was evaluated for their predictive value of SISCOM localization accuracy. Results The overall sensitivity and specificity of SISCOM in identifying an EZ was 64.8% (95%CI = 50.6–77.3) and 40.7% (95%CI = 28.1–54.3). The positive likelihood ratio and diagnostic odd ratio was 1.09 (95%CI = 0.80–1.48) and 1.26 (95%CI = 0.59–2.71), respectively. Logistic regression showed that injection latency and post-injection seizure duration did not significantly predict the probability of true positive SISCOM (p-value = 0.45 and 0.29, respectively). Conclusion Radiotracer injection latency and post-injection seizure duration were not shown to have a statistical significant impact on SISCOM performance in identifying the EZ. Significance This study demonstrates that further study of factors contributing to the performance of SISCOM in EZ identification in children is needed. [ABSTRACT FROM AUTHOR]