In Europe, Maedi-Visna disease has high prevalence rates at the individual and flock levels, respectively, and is regarded as one of the most significant infectious disease in sheep. The lack of treatment or a commercial vaccine underscores the need for accurate and reliable diagnostic tools to support control programs. Conventional methods, including ELISA and qPCR, provide useful but incomplete information due to the genetic variability of small ruminant lentiviruses (SRLVs) and the heterogeneous host immune response. In this work, third-generation sequencing was assessed as a diagnostic strategy, focusing on Oxford Nanopore Technologies amplicon sequencing of different regions of the virus genome. DNA from whole blood, PBMCs, semen, and nasal mucosa of 44 rams previously tested for Maedi-Visna virus by ELISA was used to generate amplicons of the gag, pot, and p25 genes. Sequencing showed that blood DNA was the most reliable source for SRLVs detection by Nanopore, despite the low proportion of monocytes present in this medium. Compared with conventional approaches, Nanopore sequencing reduced the proportion of false negatives observed with ELISA (42%) and qPCR (77%). These results highlight Nanopore amplicon sequencing as a promising diagnostic alternative, combining epidemiological relevance with technological innovation to enhance SRLVs detection and strengthen control strategies for sustainable disease management.