Objective We aimed to evaluate whether the internal structures of the human ear have anatomical characteristics that are sufficiently distinctive to contribute to human identification and use in a forensic context. Materials and methods After data anonymisation, a dataset containing temporal bone CT scans of 100 subjects was processed by a radiologist who was not involved in the study. Four reference images were selected for each subject. Of the original sample, 10 examinations were used for visual comparison, case by case, against the dataset of 100 patients. This visual assessment was performed independently by four observers, who evaluated the anatomical agreement using a Likert scale (1–5). Inter-observer agreement, true positive rate, positive predictive value, true negative rate, negative predictive value, false positive rate, false negative rate and positive likelihood ratio (LR+) were evaluated. Results Inter-observer agreement obtained an overall Cohen’s Kappa = 99.59%. True positive rate, positive predictive value, true negative rate and negative predictive value were all 100%. Conclusion Visual assessment of the mastoid examinations was shown to be a robust and reliable approach to identify unique osseous features and contribute to human identification. The statistical analysis indicates that regardless of the examiner’s background and training, the approach has a high degree of accuracy.