Background: Thymus is a widely used medicinal plant in food and pharmaceutical industries. Among different species of Thymus, T. vulgaris is used more than other species in therapeutic dosage forms. Objective: This study was conducted to differentiate various Thymus species by TLC fingerprint. Methods: In order to identify and differentiate various species of Thymus, TLC fingerprints of essential oil, dichloromethane, ethylacetate and n-butanol extracts of four available species named T. vulgaris, T. pubescens, T. citriodorus and T. daenensis were obtained and compared with each other. Results: The results showed that the TLC chromatograms of essential oil and n-butanol extract can not be used as differential identification for the above-mentioned species while the ethylacetate and dichloromethane extracts are more reliable to be used for TLC fingerprints. TLC chromatogram of ethylacetate extract is specific for identification of T. pubescens and T. vulgaris while T. citriodorus and T. daenensis can be best identified by their TLC fingerprints obtained from dichloromethane extract. Conclusion: For best differentiation of various Thymus species, different extracts of the plants should be used.