Allele Drop out (ADO) arising from non-amplification of one allele may produce false negative result and impact clinical management. In cancer, germline and somatic genetic analysis is being increasingly used but the prevalence, nature and implications of ADO has not been studied in any cohort. In a cohort of 290 Li Fraumeni/Li Fraumeni Like Syndrome cases undergoing TP53 genetic testing, of the 69 pathogenic mutations identified so far, 5 were initially missed and 4 were misgenotyped as homozygous mutation due to germline ADO. Of the 9 germline ADOs, 8 were sequence dependent, arising from a polymorphism (rs12951053) in the primer annealing region of exon 7. Of 35 somatic TP53 variants identified by exome sequencing in 50 oral cancer tissues registered under International Cancer Genome Consortium (ICGC), as a result of ADO, 4 were not detectable and 6 were not called as variant on Sanger Sequencing due to low peak height. High prevalence of germline and somatic ADO in the most frequently mutated cancer gene TP53, highlights the need for systematic evaluation of ADO prevalence and causes in clinically important cancer genes. False negative result for high penetrance germline mutations or actionable somatic mutations in oncogenes could have major clinical implications.