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Simple Summary: The study focuses on the genetic characteristics of the lumpy skin disease virus (LSDV) from non-vaccinated beef cattle with LSD symptoms that lived in areas that had received live-attenuated vaccines in Lopburi, Thailand. Four skin positive samples through real-time PCR underwent virus isolation on cell cultures, followed by whole genome sequencing. The phylogenetic and mutation analyses were used to compare sequences between four LSDV samples from Lopburi with the LSDV/Thailand/YST/2021 strain (original strain in Thailand). In conclusion, four LSDVs in Lopburi during 2021–2022 were recombinant strains, clade 2.5, same as the LSDV/Thailand/YST/2021 strain. However, the viruses had mutations of open reading frame (ORF) 023 with significant amino acid deletion in two samples from Chai Badan district and of the untranslated region between ORF022 and ORF023 in two samples from Lam Sothi district. Lumpy skin disease (LSD) is a viral infection that impacts the cattle industry. The most efficient approach to prevent disease involves the utilization of live-attenuated LSD vaccines (LAVs), which stands out as the most successful method. However, LAVs might be subjected to changes to their genomes during replication that increase viral infectivity or virulence. The objective of this study was to monitor alterations in the genetic characteristics of the lumpy skin disease virus (LSDV) in beef cattle following the administration of LAVs in Lopburi Province of Central Thailand. A total of four skin samples from LSD cases were collected from non-vaccinated animals that exhibited LSD clinical symptoms from two distinct districts, spanning three subdistricts within the region. The samples of cattle were analyzed using real-time PCR targeting the LSDV074 p32 gene, the virus was isolated, and the entire genome sequences were evaluated through a single nucleotide polymorphisms (SNPs) analysis, and phylogenetic trees were assembled. The investigations revealed that LSDVs from two isolates from Chai Badan district exhibited significant mutations in the open reading frame (ORF) 023 putative protein, while another two isolates from Lam Sonthi district had a change in the untranslated region (UTR). For a result, the most proficient disease diagnosis and control should be evaluated on viral genetics on a regular basis. [ABSTRACT FROM AUTHOR]