Background: Acute lymphoblastic leukemia (ALL) is a disease typically characterized by the accumulation of immature abnormal lymphoid progenitor cells (lymphoblasts) in the bone marrow, which have abnormal proliferation and differentiation. A number of acquired chromosomal abnormalities arising from translocations, deletions, duplications and inversions have been identified in 80% of childhood and 79% of adulthood ALL. 1. Objectives of Study: To determine the frequency of chromosomal abnormalities in children suffering from ALL, To evaluate structural and numerical chromosomal abnormalities in patients with ALL, To find out the frequency of Epstein Barr Virus in ALL cases. Subjects and Methods: An observational study was conducted at the Liaquat University of Medical and Health Sciences, Jamshoro and Isra University Hospital, Hyderabad. 100 diagnosed childhood ALL cases were selected through non-probability purposive sampling according to inclusion and exclusion criteria. The Blood samples were collected in bottles containing Ethylene diamine tetra acetic acid (EDTA) as an anticoagulant and were processed on automatic hematoanalyzer, Sysmex KX 21. Fixed cell suspensions prepared from diagnostic bone marrow. For routine cytogenetic analysis and FISH, samples were obtained from the diagnosed cases of acute lymphoblastic leukemia. Methods for detecting EBV infection were based on RT-PCR. The data was analyzed on SPSS version 21.0 (IBM, Corporation, USA) and Microsoft excel. The continuous variables were presented as mean ± SD and analyzed using student’s t-test. Categorical variables were analyzed by Chi-square test and results were presented as frequencies and percentages. Data was presented in tables, graphs and charts. P-value of significance was taken at ≤0.05. Results: Numerical and structural chromosomal abnormalities were noted in 69% and 60% of cases respectively (p=0.001). Chromosomal ploidy showed Diploidy and Aneuploidy in 29% and 69% of cases respectively (p=0.0001). Hyperploidy, hypoploidy and pseudoploidy were noted in 51%, 6% and 12% of cases respectively (0.001). Chromosomal structural abnormalities noted were; t (12; 21)(p13; q22) t(9;22)(q34;q11), t(8;14)(q24;q32), t(5;14)(q31;q32), t(17;19)(q22;q13), t (7;11) (q35; q13), t (1;7) (p32; q35), t (7;19) (q35; p13), t(1;19)(p13;q23), t(8;22)(q24;q11) and unknown 13%. Ph + chromosome (t (9; 22) (q34; q11) was noted in 6% of cases and EBV in 19% of total study population Conclusion: ALL cases are characterized by leukocytosis and anemia. Epstein Barr Virus was found in 19% childhood ALL in present study. Present study shows good prognostic cytogenetic abnormalities like hyperdiploidy and t (12; 21)(p13; q22) in Pakistani children with ALL and frequency of poor prognostic cytogenetic aberrations like hypoploidy and t (9; 22) (q34; q11.2) is comparable to previous studies.