Motor neuron disease (MND) is a neurodegenerative condition affecting the brain and spinal cord. It is characterized by the degeneration of primarily motor neurons, leading to muscle weakness. Patients with Amyotrophic Lateral Sclerosis (ALS) is the most common of the MNDs, combinations of both upper and lower motor neuron signs, including spasticity, hyperreflexia, and extensive plantar signs (upper motor neuron signs); and progressive muscular weakness, fasciculation, and atrophy (lower motor neuron signs), leading to fatal paralysis. It is estimated that Amyotrophic Lateral Sclerosis occurring 1.7 ~ 2.3 out of 100,000 person in worldwide. Without a cure for the condition, care focusses mainly on the maintenance of a patient’s functional abilities, allowing them to live as full a life as possible. Motor neurons control important muscle activity, such as gripping walking speaking swallowing breathing. During this study, linkage/mutational analysis and comparative genomic study was performed of motor neuron disease between humans and horses of Pakistani origin. Linkage analysis was performed for locus/gene SOD1, ALS2 and SMN1/2 in human patients while direct DNA sequencing was performed for horse gene SOD1 and ALS2. All the three genes (SOD1, ALS2 and SMN1/2) are highly involved for causing motor neuron disease in human and the ultimate objective of the study was to identify those genetic variants/mutations responsible for motor neuron disease in humans and horses. During this study, a total of 10 human families and 10 affected horses with motor neuron disease were identified from different areas of Pakistan and 5 ml blood samples were collected from affected and normal individuals for DNA extraction and estimation. In case of human pedigrees, linkage analysis was performed with the help of three STR markers to find out whether a family was linked to candidate region of the loci (SOD1, ALS2 and SMN1/2) and if a family was found to be linked with a locus/gene, subsequently the causative gene responsible for phenotype was sequenced. As a result of linkage analysis, two families (MND01, MND10) were found to be linked with human ALS2 locus/gene which was ultimately sequenced and two novel mutations (p.Ser65Ala; p.1000del) were identified in the said families (Amyotroph Lateral Scler Frontotemporal Degener 2016;17(3-4):260-265). In case of affected horses, gene SOD1 and gene ALS2 were sequenced by using DNA of affected horses. No sequence variant was found in case of gene SOD1 in any of the affected horse while one synonymous (c.1230G>A) and two missense sequence variants (c.247G>A, c.914T>G) were identified in horse gene ALS2 (in press; Journal of Animal and Plant Sciences). Although linkage analysis study was performed for locus/gene SMN responsible for motor neuron disease in human, no family was found to be lined with locus SMN. In addition, a comparative genomic analysis was performed between human and horse gene SOD1 as well as gene ALS2 to study the nucleotide similarity and protein sequence similarity. Current study has resulted in identification of two novel mutations in human gene ALS2 (Amyotroph Lateral Scler Frontotemporal Degener 2016;17(3-4):260-265) and two missense substitutions in horse gene ALS2 (in press; JAPS).)