Mucopolysaccharidoses (MPSs) are inherited lysosomal storage disorders (LSDs). MPSs are caused by excessive accumulation of mucopolysaccharides due to missing or deficiency of enzymes required for the degradation of specific macromolecules. MPS I-IV, MPS VI, MPS VII, and MPS IX are sub-types of mucopolysaccharidoses. Among these, MPS III (also known as Sanfilippo) and MPS IV (Morquio) syndromes are lethal and prevalent sub-types. This study aimed to identify causal genetic variants in cases of MPS III and MPS IV and characterize genotype-phenotype relations in Pakistan. We performed clinical, biochemical and genetic analysis using Whole Genome Sequencing (WGS) in 14 Pakistani families affected with MPS III or MPS IV. Patients were classified into MPS III by history of aggressive behaviors, dementia, clear cornea and into MPS IV by short trunk, short stature, reversed ratio of upper segment to lower segment with a short upper segment. Data analysis and variant selections were made based on segregation analysis, examination of known MPS III and MPS IV genes, gene function, gene expression, the pathogenicity of variants based on ACMG guidelines and in silico analysis. In total, 58 individuals from 14 families were included in the present study. Six families were clinically diagnosed with MPS III and eight families with MPS IV. WGS revealed variants in MPS-associated genes including NAGLU, SGSH, GALNS, GNPTG as well as the genes VWA3B, BTD, and GNPTG which have not previously associated with MPS. One family had causal variants in both GALNS and BTD. Accurate and early diagnosis of MPS in children represents a helpful step for designing therapeutic strategies to protect different organs from permanent damage. In addition, pre-natal screening and identification of genetic etiology will facilitate genetic counselling of the affected families. Identification of novel causal MPS genes might help identifying new targeted therapies to treat LSDs.