Urban visceral leishmaniasis is increasingly reported across Amazonian municipalities, where diagnostic complexity and parasite biology challenge effective surveillance and control. This study combined serological, parasitological, molecular, and sequence-based analyses of canine samples from four municipalities in the Brazilian Amazon with ultrastructural analysis of a Leishmania (L.) amazonensis reference strain. Diagnostic agreement between the Dual Path Platform rapid test (TR DPP®) and parasitological examination olymph-node aspirates was evaluated in 1,499 dogs using Cohen's kappa coefficient. Agreement was substantial (κ = 0.641; 95% CI: 0.604–0.677), although 275 dogs showed discordant results, consistent with distinct biological stages and diagnostic windows of infection. In a separate molecular subset of 130 dogs, polymerase chain reaction (PCR) targeting kinetoplast DNA (kDNA) and small subunit ribosomal DNA (SSU rDNA) revealed different detection patterns in blood and conjunctival swab samples. SSU rDNA sequencing identified a small number of nucleotide polymorphisms consistent with limited sequence variation, and sequence comparison and distance-based analyses indicated high overall similarity among the obtained sequences. Transmission electron microscopy showed lipid-associated inclusions, vacuolar compartments, and vesicle-like structures, which were described qualitatively on the basis of morphological criteria only. Because these datasets derive from complementary but distinct sources, they are presented as an integrative analytical framework rather than as evidence of mechanistic links within a single sample set. Together, the findings indicate that canine visceral leishmaniasis in Amazonian urban settings involves diagnostic discordance, matrix-dependent molecular detection, and limited SSU rDNA sequence variation, complemented by ultrastructural parasite features consistent with previous descriptions, with implications for surveillance and diagnostic strategies in endemic regions.