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Abstract Objective To highlight the reasons of culture failure in bone marrow aspirate samples sent for Cytogenetic analysis and to identify the associated parameters causing this impact. Methodology This is a retrospective cross-sectional study conducted in the Clinical and Molecular Cytogenetics Laboratory of NIBD Hospital, Karachi, Pakistan. The rates of culture failure are assessed from the year 2017–2020 along with their reasons. Bone Marrow aspirate samples of patients with hematological malignancies were cultured for chromosomal analysis, both at the time of diagnosis or relapse. Statistical analysis was performed using SPSS version 25. Results A total of 1061 bone marrow aspirate samples were assessed for cytogenetic culture failures from the duration of 2017 to 2020. Ratio of males was predominantly higher i.e. 62.7% than female 37.3% with Mean ± SD age was 36.78 ± 18.94. Frequency of culture failure in the year 2020 was relatively high 20% as compared to the preceding years i.e. 8% in 2017, 6% in 2018, 7% in 2019. However, the patients were diagnosed with the following hematological malignancies; ALL 23%, CML 17.1%, AML 16.5% and AA 12.5%. Among the reasons of culture failure, cytogenetic analysis of patients with on-going chemo resulted in significant culture failures with p-value