The Bcr-Abl and Lyn protein tyrosine kinases have been separately linked to the emergence of imatinib resistance in patients with chronic myelogenous leukemia. We have developed fluorescent sensors for these kinases that are enzymatically and photophysically distinct, allowing us to simultaneously, yet separately, visualize the tyrosine kinase activities of both Abl and Lyn. Multicolor monitoring revealed that an imatinib-resistant cell line (MYL-R) displays a remarkable 13-fold enhancement in Lyn kinase activity relative to its imatinib-sensitive counterpart (MYL). By contrast, both cell lines display nearly identical Abl activities. The upregulation of Lyn kinase phosphotransferase activity in MYL-R cells is linked to an overexpression of the Lyn B isoform. Furthermore, MYL-R cells possess a 4-fold higher level of activated Lyn and 5-fold lower level of autoinhibited Lyn than MYL cells. Finally, studies with an activating SH2 ligand revealed that Lyn from imatinib-resistant MYL-R cells is primed and active, whereas Lyn from imatinib-sensitive cells is dependent upon phosphorylated SH2 ligands for activity.