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<i>Knotted1</i>-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As <i>Prunus</i> species include important fruit tree crops of Italy, an exhaustive investigation of <i>KNOX</i> genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated <i>KNOX</i> transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in <i>Prunus</i> spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach <i>KNOX</i> transgenes. Based on ten <i>Prunus</i> spp., <i>KNOX</i> proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of <i>Prunus</i> from <i>Cerasus</i> subgenera, and that of <i>Armeniaca</i> from the other sections within <i>Prunus</i>. Cytokinin (CK) cis-elements occurred in peach and almond <i>KNOX</i> promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by <i>KNOX</i> expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified <i>KNOX</i> transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6–0.6%) with the genetic construct, with <i>35S:GFP</i> being more stable than <i>35S:KNOPE1</i> lines, which showed leaf modification typical of <i>KNOX</i> overexpression.