We assessed the stability of transgene expression in 79 transgenic lines (i.e., transformation events) of hybrid poplars during several years of field trials. The transgenic lines were comprised of 40 lines of hybrid cottonwoods (P. trichocarpa × P. deltoides) that were grown at three field sites, and 39 lines of hybrid aspens (section Leuce, P. alba × P. tremula) that were grown at a single field site. All the lines were transformed with a binary construct that included two genes that confer tolerance to glyphosate (GOX and CP4), a gene encoding resistance to the antibiotic kanamycin (nptII), and a visible marker gene (GUS). Agrobacterium tumefaciens was used for transformation; callogenesis and organogenesis occurred under kanamycin selection. In addition to repeated applications of herbicide to test stability of transgene expression, for the first time, we challenged ramets of 40 lines that had not previously been tested for herbicide resistance in their fourth season of vegetative growth. We report on the stability of herbicide resistance and GUS expression and evidence for somaclonal variation in growth and leaf morphology.