The Colorado potato beetle, Leptinotarsa decemlineata, is a major holarctic pest of solanaceous crops. Presumably, this insect spread from Solanum species in central America to the Mexican plateau, and this was followed by multiple invasions of North America and Europe. Attempts are being made to control this beetle by using a genetically modified spiroplasma that occurs naturally in its gut. In the current study, spiroplasmas isolated from beetles collected in North America and Poland exhibited serologic (spiroplasma motility inhibition test) and genomic (restriction fragment length polymorphism) profiles that suggest that there were multiple spiroplasma introductions. Two serovars were identified; one is found in northern North America and at high elevations in Poland, and the other is found in southern North America and at low elevations in Poland. The patterns of genovar distribution coincide with the serovar patterns. The existence of such biovars—intraspecific taxal units reflected by serologic and genomic differences—should be taken into consideration when taxonomies are developed and strains are chosen for biocontrol