Bacterial spot caused by Xanthomonas spp. is the second most important bacterial disease after bacterial wilt of tomato and pepper in Taiwan. To determine the species composition of the Xanthomonas population over 27 years (1989 to 2016) across the country, a large collections of strains from tomato (n = 292) and pepper (n = 198) were examined. In the 1989 to 1999 population, all strains (n = 147) from pepper and 95% strains (n = 198) from tomato were Xanthomonas euvesicatoria. The remaining 5% of strains from tomato were X. vesicatoria. In a 2000 to 2009 population from tomato (n = 36), 22% of the strains were X. perforans and the remaining 78% strains were X. euvesicatoria. In the 2010 to 2016 population, 92% of the strains (n = 50) from pepper were still X. euvesicatoria and the remaining 8% of the strains were X. perforans; however, 99% (n = 58) of the strains from tomato were X. perforans. All of the evaluated (n = 25) strains of X. euvesicatoria collected during 1990 to 2006 were tomato race T1. Four pepper races (P1, P2, P7, and P8) were identified in the X. euvesicatoria population. The strains of X. vesicatoria collected during 1989 to 1999 (n = 8) were tomato race T2 and strains of X. perforans from tomato collected during 2010 to 2016 (n = 12) were race T4 (83%) and race T3 (17%). Four strains of X. perforans from pepper were race T4. All of the strains of X. vesicatoria and X. perforans caused a hypersensitive response in all pepper differentials. Biochemical characterization of representative strains (n = 48) showed that strains of X. euvesicatoria were negative on and amylolytic test and positive on lipase and oxidative-fermentative (OF) tests. The strains of X. vesicatoria were positive on amylolytic and OF tests and were negative on the lipase test. All X. perforans strains showed positive reactions on three tests. Evaluation of the same 48 strains for the sensitivity to copper sulfate (50, 100, 200, 300, and 400 mg liter-1) revealed that the majority of X. euvesicatoria (86%) and X. perforans (94%) strains in the 2010 to 2016 population were tolerant to copper sulfate. The findings suggest that management strategies and breeding programs should consider the new X. perforans species and their new races. The increased number of copper-sulfate-tolerant strains in the 2010 to 2016 population further shows the need for alternative options to copper for managing bacterial spot of tomato and pepper.