To enforce timber import laws and perform timber species identification, the identity of the botanical species must be well-defined. Since the Sapotaceae family is known as a taxonomically challenging family, we focus in this study on the four most valuable Sapotaceae timber species from tropical Africa: Autranella congolensis (De Wild.) A.Chev., Baillonella toxisperma Pierre, Tieghemella africana Pierre and Tieghemella heckelii (A.Chev.) Pierre ex Dubard. The wood anatomical characteristic fiber lumen fraction and Direct Analysis in Real Time—Time of Flight Mass Spectrometry (DART-TOFMS) were used to differentiate the four species and to make inferences on species delineation and taxonomic identity.