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Urban visceral leishmaniasis is intensifying across Amazonian cities, where ecological gradients, diagnostic divergence, and parasite adaptation complicate surveillance. We applied a One Health translational framework integrating diagnostic performance (TR DPP®, parasitology, kDNA, SSU rDNA), parasite microdiversity, ultrastructural remodel-ing, and extracellular vesicle (EV) biology across four municipalities. Among 1,499 dogs, infection burden was high and spatially heterogeneous. Agreement between TR DPP® and parasitology was substantial (κ = 0.6483), with 269 discordant outcomes reflecting distinct biological windows of infection. Blood and conjunctival swabs provided comple-mentary molecular detection, consistent with tissue specific parasitism. SSU rDNA se-quencing revealed low level polymorphisms compatible with intraregional microdiversity, while phylogenetic reconstruction confirmed genetic stability. Ultrastructural analyses identified lipid associated morphotypes and vesicle related structures supporting meta-bolic plasticity. EV release increased under thermal stress, indicating stress responsive ve-siculation with potential implications for immune modulation and diagnostic divergence. These integrated findings clarify how diagnostics, microdiversity, ultrastructure, and EV mediated communication converge to shape urban leishmaniasis dynamics and sup-port improved surveillance strategies for Amazonian endemic areas.