In 2003, symptoms of Pierce Disease (PD) were observed in two vineyards established in two different localities in San Jose province (Santa Ana and La Uruca) and in La Garita, Alajuela province. Twenty four symptomatic plants (15 from Santa Ana, five from La Uruca and four from La Garita) were analyzed by DAS-ELISA using antibodies against Xylella fastidiosa. Sixteen plants (nine from Santa Ana, five from La Uruca, and two from La Garita) were positives for the bacterium. Isolation attempts were made from petioles of these plants, and seven isolates were obtained in liquid PW. Aliquots of liquid media were inoculated on solid PW. Ten days after inoculation, circular and convex colonies, with entire margin, smooth and opalescent, of 0.5-1mm in diameter were observed. From each isolate three colonies were selected and considered as different clones. Every clone was analyzed by DAS-ELISA with positive results. Clones were Gram negative, catalase positive and oxidase negative. The bacteria were rod shaped, and showed a typically rippled cell wall. DNA of each clone was extracted and used as template in PCR with primer's 272-1/272-2 and RST31/RST33; products of 500pb and 733pb were obtained, respectively. These results confirmed the presence of X fastidiosa causing PD in grapevine plants in different vineyards in Costa Rica.