The distribution of lethal wilt, a severe disease of oil palm, is spreading throughout South America. An incidence of about 30% was recorded in four commercial fields in Colombia. In this study, phytoplasmas were detected in symptomatic oil palm by using specific primers, based on 16S ribosomal DNA (rDNA) sequences, in nested polymerase chain reaction assays. The phytoplasmas were then identified as ‘Candidatus Phytoplasma asteris’, ribosomal subgroup 16SrI-B, through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing. Cloning and sequencing of 16S rDNA from selected strains, together with phylogenetic analysis, confirmed the classification. Moreover, collective RFLP characterization of the groEL, amp, and rp genes, together with sequence data, distinguished the aster yellows strain detected in Colombian oil-palm samples from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.