In E × B sources with different degrees of magnetization between electrons and ions, collisionless Simon¬–Hoh instability (CSHI) is prone to be induced that may result in long-wavelength and low-frequency plasma oscillation along the azimuthal direction known as the rotating spoke. Recent studies have shown that the inertial response of unmagnetized ions is a key factor that affects the rotating spoke frequency, and in-depth studies on the correlation between the ion mass and spoke frequency have been conducted for various types of E × B sources (e.g., Hall thruster, magnetron, and Penning sources). In the case of the E × B Penning source where the generation of desired ion species is maximized through molecular gas discharge, the intrinsic characteristics of the instability dependent on the ion mass inevitably require consideration of the coexistence of various ion species. In this work, we experimentally studied the spoke in the E × B Penning source with two-ion-species plasma. By adjusting the mixture ratio of argon and krypton gases, we investigated the changes in instability frequency with electrical diagnostics. Two frequency peaks were clearly observed, with the peaks of lower and higher frequency corresponding to mode number 2 and 3, respectively. The frequencies of each peak shifted to higher values when the argon content in the gas mixture was increased. Finally, the theoretical scaling of the spoke suggests that the changes in the frequencies were proportional to the inverse square root of the effective ion mass.