Atractylodis lancea (Cangzhu) and Atractylodes macrocephala (Baizhu) are two traditional Chinese medicines with complex chemical compositions. However, a comprehensive comparative analysis of their constituents remains lacking. In this work, an ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap tandem mass spectrometry (UPLC-Q-Orbitrap-MS/MS) method was developed for the rapid characterization and differentiation of chemical components in Cangzhu and Baizhu. Chromatographic separation was achieved on a C18 column with gradient elution, and compounds were detected using electrospray ionization in both positive and negative modes. By analyzing characteristic fragmentation patterns and neutral losses, a total of 111 compounds were tentatively identified, including 14 common components (present in both species) and 97 differential components (unique to one species). This study presents the first comprehensive comparative chemical profiling of the two Atractylodes species, offering valuable references for pharmacodynamic material basis studies and quality control.