This study aims to improve the ILTV genotyping assays by amplifying an expanded region of the viral genome through multiplex PCR and adopting the MinION sequencing technology to increase the depth of coverage on informative SNP sites to provide accurate discrimination between ILTV strains. The Unique short (Us) region of the ILTV genome was targeted for multiplex PCR development. Sixteen primer pairs were designed, each capable of amplifying approximately a 1kb fragment within the Us ILTV genome region. The sequence analysis of the US sequences of known ILTV isolates were assigned to corresponding genotypes as characterized by full genome analysis and multi-allelic Sanger sequencing assay. Applying the multiplex PCR MinION sequencing assay to clinical samples, demonstrated successful genotyping, and increased the depth of coverage of the sequences allowed to identify four SNPs within the Us sequences not previously identified among currently circulating genotype VI virus.