Thyroglossal duct cysts (TDCs) with ambiguous epithelial lining are the most common midline cervical anomaly encountered in children. To histopathologically study the epithelial lining of 32 thyroglossal duct cysts in relation to their locations. A total of 32 cases of thyroglossal duct cysts were collected for this study. The samples were classified into 3 groups as follows: (1) above the hyoid bone; (2) at the level of hyoid bone; (3) below the hyoid bone. All samples were H&E stained. Seventeen TDCs (53 %) were found above the hyoid bone, 6 (18.8 %) were found at the level of hyoid bone and 9 (28.1 %) were found below the hyoid bone. Of the 32 total cases, 6 (18.8 %) were lined with stratified squamous epithelium (SSE), 17 (53 %) were lined with pseudostratified ciliated epithelium (PSCE), 3 (9.4 %) were lined with stratified cuboidal epithelium (SCE), and 6 (18.8 %) exhibited both SSE and PSCE. Pseudostratified ciliated epithelium was dominant in the region superior to the hyoid bone, whereas SCE was detected only in TDCs at the level of hyoid bone. These differences were statistically significant (P = 0.0001). Different types of epithelial lining were detected in the study samples of TDCs. A statistically significant correlation was found between the type of epithelium detected and the location of the TDC at the time of diagnosis.