Glandular chemical defence relying on the action of salicylaldehyde is characteristic for
Chrysomela
leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in
Chrysomela
species. We here identified and characterized SAO of
Phratora vitellinae
, the only species outside the genus
Chrysomela
that produce salicylaldehyde as a defensive compound. Although
Chrysomela
and
Phratora
are not closest relatives, their SAOs share glucose–methanol–choline oxidoreductase (GMC) affiliation, a specific GMC
i
subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both
Chrysomela
and
Phratora
. Closely related species of
Chrysomela
and
P. vitellinae
use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.