Aims: We have examined the intraspecific diversity of a collection of 63 Spanish strains of Erwinia amylovora, isolated from 1995 to 2001, to determine whether or not they could be grouped based on phenotypic or genotypic criteria and to investigate the sources of inoculum for fire blight dissemination in Spain. Methods and Results: Several biochemical and molecular techniques, such as miniaturized API 20E, API 50CH, ATB G-5 and API-ZYM tests, BIOLOG metabolic fingerprinting, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), minisatellite-primed PCR (MSP-PCR), random amplified polymorphic DNA (RAPD) analyses and AFLP were used. We report the first identification in Spain of the PFGE pattern Pt1, already described in other European countries, together with Pt3 and Pt4 patterns. Moreover, PFGE, together with MSP-PCR, RAPD analyses and AFLP are, until now, the only techniques that have provided information about the possible infection sources and relationships between the different foci in Spain, with AFLP being the most discriminative. Conclusions: These techniques have allowed grouping of Spanish strains by their geographical origin. Significance and Impact of the Study: Our results support the hypothesis that some fire blight outbreaks have been caused by the introduction in Spain of infected plant material, or other inoculum sources from different European countries.