Pacritinib, potent inhibitor of Janus kinase 2 (JAK2), JAK2V617F, and fms-like receptor tyrosine kinase 3, is in Phase III development in myelofibrosis. Among type 1 inhibitors, pacritinib shows a lack of myelosuppression at doses that both inhibit JAK2/signal transducer and activator of transcription 3 pathway and demonstrate clinical efficacy. To elucidate these mechanisms and identify other disease targets, a kinome analysis screened 439 recombinant kinases at 100 nM pacritinib concentration. For kinases with >50% inhibition, pacritinib was titrated from 1 to 100 nM. JAK2, JAK2V617F, FLT3, colony-stimulating factor 1 receptor, and interleukin-1 receptor-associated kinase 1 achieved half-maximal inhibitory concentrations <50 nM. Pacritinib did not inhibit JAK1 (82% control at 100 nM). Lack of myelosuppression may stem from inhibiting JAK2 without affecting JAK1 and reducing hematopoietic inhibitory cytokines by suppressing interleukin-1 receptor-associated kinase 1 or colony-stimulating factor 1 receptor. The pacritinib kinome suggests therapeutic utility in acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, solid tumors, and inflammatory conditions.