The aim of this study is to compare the two-dimensional transesophageal echocardiography (2D-TEE) with multi-slice computed tomography (MSCT) and with autopsied material to evaluate the ability and precision of the imaging methods for the detection and assessment of septal pouch (SP). One hundred and fifty patients that underwent both 2D-TEE and MSCT and 50 autopsied human hearts were investigated. In MSCT, the interatrial septum was classified as a left SP in 37.3%, right SP in 3.3%, and patent foramen ovale (PFO) channel in 3.3%. In 2D-TEE, the interatrial septum was classified as a left SP in 39.3%, right SP in 11.3%, double SP in 4.7%, and PFO channel in 2.0%. The weighted kappa coefficient between MSCT and 2D-TEE in assessing the septum morphology was 0.59. The prevalence of the left SP is lower when it is evaluated by MSCT or 2D-TEE than by anatomical study, but this difference is insignificant (37.3% vs. 44.0%, p = 0.40, and 39.3% vs. 44.0%, p = 0.56, respectively). The prevalence of left SPs is lower when detected by MSCT or 2D-TEE than during autopsy, but the difference is statistically insignificant (37.3% vs. 44.0%, p = 0.40, and 39.3% vs. 44.0%, p = 0.56, respectively). Both 2D-TEE and MSCT are comparable techniques for detecting left-sided SPs with the insignificant lower prevalence of detecting left-sided SPs compared to autopsied material. However, TEE with the contrast seems to be superior over MSCT due to the possibility of unambiguous PFO channel detection. Therefore, TEE with contrast should be preferred over MSCT in the identification of left-sided SPs.