The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in signaling for a diverse range of cytokines and growth factors in mammals, controlling various cellular processes. Within the STAT family, the STAT5B protein is implicated in a spectrum of hematopoietic diseases. Dysregulation of STAT5B phosphorylation is associated with numerous conditions, including clonal hematopoietic stem cell disorders like Myeloproliferative Neoplasms (MPNs). Understanding the intricate phosphorylation mechanisms of STAT5B is crucial for deciphering the pathogenesis of diseases linked to JAK-STAT pathway dysregulation. This study delves into the impact of phosphorylation on STAT5B conformational changes, employing over 400 ns explicit molecular dynamics (MD) simulations and comprehensive computational analyses, including the machine-learning-based bound ion coordination detector, HIT2. The results indicate that phosphorylation induces a redistribution of electrostatic charges and alters hydrogen bond formations in antiparallel structures. Moreover, phosphorylated parallel STAT5B dimers exhibit significantly negative surface changes and dense electric field lines, augmenting interactions within the STAT5B dimers. These discoveries shed light on the molecular mechanisms governing STAT5B and present potential therapeutic targets for hematopoietic diseases involving aberrant STAT5B activation.