Small Ruminant Lentiviruses (SRLV) include at least 4 viral highly divergent genotypes. Genotypes A and B are widely distributed and genotypes C and E have been recognized in restricted geographic areas. New phylogroups have been identified targeting conserved regions. However, this approach suffers from the potential risk to misamplify highly divergent strains. Pathogenic strains are easily adapted to fibroblastic cells, but non-pathogenic strains isolation may require a different approach. 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) during regular slaughter in two different slaughterhouse 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 the concentrated supernatant with the highest value of RTA and reverse transcribed as double stranded cDNA. The samples were sequenced with a Next Generation Sequencing (NGS) approach, using Nextera XT protocol and Illumina MiSeq platform. Data analysis was conducted following two parallel approaches, resequencing and de-novo assembling, with Geneious and Velvet softwares respectively. Twenty two spleen explants allowed viral isolation and full genome sequencing. All the samples showed high variability of in vitro behavior. Only 13 RTA positive samples showed CPE on overgrowing fibroblastic-like cells. A positive correlation was observed between the phylogroup A8 and the absence of CPE in culture while all strains belonging to subtype B1 and B2 were fusogenic in vitro. Moreover two new subtypes (A18 and A19) were described. In conclusion the proposed method proved to be a fast and economically feasible approach for viral full genome characterization as well as for biological and in vitro properties study. Despite the limited sampling area it allows to detect both pathogenic subgroup B1 and B2, putative non pathogenic A8 in goats, two novel A subgroups. The success in virus isolation with this method is based on the association of three features: the choice of the spleen as target tissue for virus isolation, the use of RTA as sensitive method to detect viral replication and the use of a high throughput technology for genome sequencing.