We evaluated a microcolony method for the detection and identification of beer-spoilage lactic acid bacteria (LAB). In this approach, bacterial cells were trapped on a polycarbonate membrane filter and cultured on ABD medium, a medium that allows highly specific detection of beer-spoilage LAB strains. After short-time incubation, viable cells forming microcolonies were stained with carboxyfluorescein diacetate (CFDA) and counted with muFinder Inspection System. In our study, we first investigated the growth behavior of various beer-spoilage LAB by traditional culture method, and Lactobacillus lindneri and several L. paracollinoides strains were selected as slow growers on ABD medium. Then the detection speeds were evaluated by microcolony method, using these slowly growing strains. As a result, all of the slowly growing beer-spoilage LAB strains were detected within 3 days of incubation. The specificity of this method was found to be exceptionally high and even discriminated intra-species differences in beer-spoilage ability of LAB strains upon detection. These results indicate that our microcolony approach allows rapid and specific detection of beer-spoilage LAB strains with inexpensive CFDA staining. For further confirmation of species status of detected strains, subsequent treatment with species-specific fluorescence in situ hybridization (FISH) probes was shown as effective for identifying the CFDA-detected microcolonies to the species level. In addition, no false-positive results arising from noise signals were recognized for CFDA staining and FISH methods. Taken together, the developed microcolony method was demonstrated as a rapid and highly specific countermeasure against beer-spoilage LAB, and compared favorably with the conventional culture methods.