In this study, the traditional physiological and biochemical method, a VITEK automatic biochemical analyzer, the analytical profile index (API) biochemical identification system, real-time fluorescence quantitative polymerase chain reaction (real-time PCR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were used comparatively to identify the species of Pediococcus spp. in food. The results showed that the traditional physiological and biochemical method was the most accurate but time-consuming and labor-intensive; the VITEK automatic biochemical analyzer gave unstable results as the database states that Pediococcus acidilactici is negative for amygdalin reaction, whereas some strains actually test positive; the API biochemical identification system yielded accurate results at 24 h of culture, while deviations occurred when the culture period was extended to 48 h; real-time PCR was time-saving, accurate, and suitable for popularization; the MALDI-TOF MS method had the shortest operation time but high equipment requirements, making it difficult to popularize in primary-level institutions. In summary, the traditional physiological and biochemical method, real-time PCR, and MALDI-TOF MS can all serve as standard methods for the identification of Pediococcus spp. in food. The results of this study can provide a technical basis for the formulation of relevant national standards.