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A crucial part of a gyroklystron is an electron beam. For the efficient operation of the gyroklystron, effective interaction between the radio frequency wave and the electron beam is necessary. Although the manufacturing and assembly of the gyroklystron are complicated, during the assembly process of various components of the gyroklystron, there is some possibility that the components may be misaligned from their actual positions. The operation of a gyroklystron is very sensitive if its components are misaligned. Electron beam misalignment is one type of such misalignment. This paper studies the effect of an off-axis electron beam on gyroklystron performance. Due to misalignment, the electron beam radius will vary in its position as the electron beam gyrates. First, the nonlinear single-mode analysis describing the beam–wave interaction process within the gyroklystron amplifier is discussed. As reported in the literature, an experimental, two-cavity 35 GHz fundamental harmonic gyroklystron amplifier has been used to conduct the present study. The results obtained from the analytically developed approach are benchmarked by the results of the experimental device. Subsequently, the analysis developed has been extended to investigate the impact of the misaligned electron beam on the gyroklystron performance characteristics. The study shows that efficiency, output power and gain decrease with misalignment, whereas bandwidth does not change due to such misalignments. These findings demonstrate the critical role that the misaligned electron beam plays in the operation of a gyroklystron amplifier. [ABSTRACT FROM AUTHOR]