The pathogenic bacterium Pseudomonas syrinage pv. actinidiae causes bacterial canker disease, which is the most damaging disease afflicting kiwifruit (Actinidia chinensis). The most prevalent strain is biovar 3 (Psa3), which is highly pathogenic. Therefore, Psa3-resistant varieties of kiwifruit are urgently needed. A previous study reported that Actinidia rufa, a wild Japanese species related to kiwifruit, is highly resistant to Psa3. Genome-wide DNA markers may be useful to locate the loci that confer Psa3 resistance. Therefore, in the present study, we used published kiwifruit genome sequence information to design 1,101 simple sequence repeat (SSR) markers covering the entire kiwifruit genome in silico. We next examined DNA polymorphisms in more than 1,000 polymerase chain reaction products for potential use as SSR markers among A. rufa Fuchu and A. chinensis FCM1, a pollen-providing parent Fuchu A. chinensis male 1, and other kiwifruit cultivars in order to develop Psa3 resistant kiwifruit cultivars. We finally obtained 351 polymorphic markers, in the entire kiwifruit genome, which can be applied to produce next-generation kiwifruit cultivars, including Psa3-resistant cultivars.