Small Ruminant Lentiviruses (SRLV) includes at least 4 genotypes and several subtypes. The viral heterogeneity can influence the viral-host interaction, the cell tropism, the biological properties and the diagnostic results. The major in vivo tropism of SRLV is for monocyte/macrophages and dendritic cells in fibroblast-like cells in case of high virulent strians. Several viral strains are characterized by low pathogenic effects and show a limited ability to replicate in fibroblast cells. Keeping in mind the heterogeneity of in vitro properties of SRLV we developed a fast and effective method for SRLV full genome characterization from spleen explants after cell culture isolation. Blood and spleen samples were collected from 42 adult animals (16 sheep and 26 goats) in two different slaughterhouses in Northern Italy. Blood samples were tested with a commercial kit for antibody screening and genotyping. Spleen derived macrophage culture was maintained over 5 passages and monitored for the appearance of cytopathic effect and Reverse Transcriptase Activity (RTA). Total RNA was extracted from supernatant and reverse transcribed as double stranded cDNA for Next Generation Sequencing on Illumina MiSeq. Data analysis was conducted by resequencing and de-novo assembling approaches. 22 spleen explants allowed viral isolation and full genome characterization. Only 13 RTA positive samples showed CPE. A positive correlation was observed between the phylogroup A8 and the absence of CPE. All strains belonging to subtype B1, B2, and A9 were fusogenic in vitro. Despite the limited sampling area it allows to detect both pathogenic subgroup B1 and B2, putative non pathogenic A8 in goats, an additional A9 isolate in sheep and a novel A subgroup in goat. In conclusion the proposed method proved to be a fast and economically feasible approach for viral full genome characterization as well as for biological