In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied. Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample. Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences). Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related. In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia. This indicated that these isolates probably constituted a new species. An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize. More isolates of this group were subsequently obtained from field-grown maize and teosinte plants. It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize.