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For fast and cost-efficient identification of bacteria we tested the Biotyper system (Bruker Daltonics) to detect Erwinia amylovora and other plant-associated bacteria by protein profiles from whole cells. Strains could also be identified by direct transfer of cells to the sample target, and the signals were compared to preceding protein extraction with formic acid. The identification scores obtained for extracted proteins were higher and more reliable than those of directly applied agar cultured cells. For verification of MALDI-TOF identification and for detection of several Erwinia species in mixed cultures we applied PCR primer sets designed from specific chromosomal sequence regions such as the gene cluster for biosynthesis of capsular EPS or the nucleotide sequences flanked by genes pstS and glmS. The novel primers readily detected E. amylovora fruit tree and raspberry strains, E. pyrifoliae, E. billingiae and E. tasmaniensis. The identification obtained by PCR agreed with the MALDI-TOF MS results. While PCR analysis is also applicable for detection of bacteria in mixed cultures obtained from plant samples, MALDI-TOF analysis identifies essentially cells from single colonies applied from agar plates or cultured in liquid medium. Both methods were combined for identification of bacteria in field samples and from artificially inoculated plant material.