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Background: Antibiotics are commonly used in animal husbandry for medicinal, prophylactic and growth promotion purposes. Method: The prevalence of three groups of antibiotics; namely Gentamycin, Streptomycin and sulfonamides was studied in fresh milk available in five different study areas; namely Budhanilkantha, Dharmasthali, Tokha, Nepaltar and Gaushala from Kathmandu Valley of Nepal. The milk samples (n=100) were collected from local dairy collection centers of Kathmandu valley. Qualitative and semi-quantitative analysis with rapid screening kits revealed that 6% (n=6) samples were positive for antibiotic residues in the fresh milk for gentamycin, streptomycin and sulfonamide groups. Total 5% milk samples contained unidentified antibiotics residues. Sulfonamides residues were found to be highest 3% (n=3) followed by gentamycin 2% (n=2) and streptomycin 1% (n=1) with no any milk sample with multiple residues. Total 2 samples were positive in Dharmasthali, 2 in Gaushala, 1 in Nepaltar, 1 in Tokha and no positive samples from Budhanilkantha area. There was no statistical significant difference between study areas and commonly used antibiotics residues in milk samples (P>0.05). Conclusion: The antibiotic residues were found above the maximum residue limits that presented serious threat to consumer health and raised a serious concern regarding the implementation and monitoring of international regulations. [ABSTRACT FROM AUTHOR]