Psychrotrophic bacteria in raw milk produce heat-resistant extracellular proteases, causing spoilage in ultrahigh temperature (UHT) treated milk and milk products. Rapid and reliable identification systems are required for screening of spoilage microbes in raw milk upon delivery to the processing precinct. This study aimed to compare four commercial bacterial identification systems with 16S rRNA sequencing for identification of heat-resistant protease producing bacteria in raw milk. The comparison was based on the typability, discrimination power [i.e. Simpson’s index of diversity (SID score)], reproducibility and speed of analysis. The identification accuracy of Gram negative bacteria (GNB) by five systems to the species level was: 16S rRNA gene sequencing (100.0%)>Biolog (86.8%)>MALDI-TOF MS (63.2%)>API (60.5%)>Microbact (57.9%). Gram positive bacilli (GPB) were identified by 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API with accuracies at the species level of 100.0%, 85.0%, 95.0% and 90.0%, respectively. 16S rRNA gene sequencing and phylogenetic analysis discriminated Pseudomonas fluorescens, Pseudomonas syringae, Hafnia alvei, Bacillus cereus, Bacillus pumilus and Bacillus licheniformis to the subspecies level. The SID scores were 0.966, 0.711, 0.496, 0.472, and 0.140, for 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API and Microbact, respectively. The rapidity of each assay is in the following order: MALDI-TOF MS>16S rRNA gene sequencing>Biolog> Microbact>API. Thus, 16S rRNA gene sequencing and Biolog are suitable for identification of GNB, while 16S rRNA gene sequencing, MALDI-TOF MS and API appear to be suitable for identification of GPB isolated from milk.