The predatory mite Neoseiulus barkeri (Hughes) is a good biological control agent for many small sucking pests. We aimed to determine whether rearing long term on alternative prey versus target prey species affected the performance of N. barkeri. Therefore, we investigated the prey preference, life tables, and population parameters of N. barkeri between alternative prey Tyrophagus putrescentiae (Schrank) and three species of spider mites, Tetranychus urticae Koch, Panonychus citri (McGregor), and Eotetranychus kankitus Ehara. We found that N. barkeri preferred the tetranychid mites to the alternative prey. Between the tetranychid mites, the predator consumed more P. citri and E. kankitus than T. urticae. When reared on T. urticae, the total developmental time and longevity of N. barkeri were the longest, whereas the intrinsic rate of increase was the lowest, indicating that the biotic fitness of predatory mite preyed on target of T. urticae was higher than on alternative prey of T. putrescentiae. However, total developmental time, longevity, and fecundity did not differ between N. barkeri reared on T. putrescentiae and P. citri, although these parameters were higher than those for mites reared on E. kankitus, indicating that the predatory mite reared on T. putrescentiae may not be affected to control P. citri, and that coexistence of P. citri and E. kankitus may enhance the control efficiency of N. barkeri. Altogether, our results demonstrated that long-term feeding on the alternative prey T. putrescentiae did not affect the performance of the predatory mite N. barkeri on various target spider mites.