Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.Avian intestinal spirochetosis (AIS) is a disease complex that affects commercial laying and meat breeder chickens, characterized by the colonization of the ceca by members of one or more species of anaerobic intestinal spirochetes of the genus Brachyspira (formerly Serpulina) (11,14,36). The condition is associated with delayed or reduced egg production and a chronic diarrhea in adult birds that can result in wet litter and fecal staining of eggshells. Moreover, reduced performance has been reported for broiler chicks hatched from eggs of breeder chickens infected with Brachyspira spp. (7,33). AIS was first clearly described in the 1980s, in both The Netherlands (5, 7, 8) and the United Kingdom (10), and subsequently has been reported in the United States (39, 43), Australia (24,35), and a number of other European countries (2, 3). The nonspecific nature of the disease and the lack of availability of simple and specific diagnostic tests for these anaerobic spirochetes from chickens may have resulted in the condition being greatly underreported (14).Studies using multilocus enzyme electrophoresis showed that intestinal spirochetes from chickens could be divided into at least six species groupings (25). These included the three species currently considered to be pathogenic to chickens, Brachys...