Multilocus enzyme electrophoresis was employed to estimate chromosomal genotypic diversity and relationships among 131 isolates of the non-motile Salmonella biotypes Gallinarum and Pullorum (serotype 1, 9, 12:-:-) that cause fowl typhoid and pullorum disease, respectively. Thirteen electrophoretic types (ETs), marking clones, were distinguished, and construction of a neighbour-joining phylogenetic tree revealed three lineages: one consisted of five ETs of Gallinarum, a second included seven ETs of Pullorum, and a third was represented by a single ET (Ga/Pu 1) that is intermediate between those of the other two lineages in both multilocus enzyme genotype and biochemical properties. Enzyme genotype analysis and comparative nucleotide sequencing of the phase 1 flagellin gene (fliC), the hook-associated protein 1 gene (flgK), and the 6-phosphogluconate dehydrogenase gene (gnd) identified serotype Enteritidis (1, 9, 12:g, m:-) as a close relative of the non-motile salmonellae. In most strains of biotype Gallinarum, the fliC gene is complete, intact and identical in sequence to that of Enteritidis, but isolates of three ETs had a stop codon at position 495. The fliC sequences of the ETs of Pullorum differed from that of Enteritidis in having non-synonymous changes in either two or three codons and a synonymous change in one codon. The sharing of distinctive alleles at three metabolic enzyme loci and a stop codon in flgK indicates that the non-motile salmonellae are monophyletic and that their most recent common ancestor was non-motile. Since diverging from that ancestor, the Pullorum lineage has evolved more rapidly than the Gallinarum and Ga/Pu 1 lineages.