BACKGROUND:  Multi-drug Resistant (MDR) bacteria could lead to treatment failure of infectious diseases and could be transferred by non-potable water. Few studies have investigated occurrence of Antibiotic Resistance Genes (ARGs) among bacteria including Aminoglycoside Modifying Genes (AMGs)from Drinking Water Distribution Systems (DWDS) in Nigeria. Here, we aimed at characterization of AMGs from DWDS from selected states in southwestern Nigeria.METHODS: One hundred and eighty one (181) MDR bacteria that had been previously characterized using 16S rDNA and showed resistance to at least one aminoglycoside antibiotic were selected from treated and untreated six water distribution systems in southwestern Nigeria. MDR bacteria were PCR genotyped for three AMGs:aph (3´´)c, ant (3´´)band aph(6)-1dd.RESULTS: Out of 181 MDR bacteria genotyped, 69(38.12%) tested positive for at least one of the genotyped AMGs. Highest (50, 27.62%) detected gene was ant (3”)cfollowed by aph (3")c(33, 18.23%). Combination of aph(3")c  and ant (3")bin a single bacteria was observed as the highest (14, 7.73%) among the detected gene combination. Alcaligenes sp showed the highest (10/20) occurrence of ant (3")bwhile aph(3")c was the highest detected among Proteus sp (11/22). Other bacteria that showed the presence of AMGs include: Acinetobacter, Aeromonas, Bordetella, Brevundimonas, Chromobacterium, Klebsiella, Leucobacter, Morganella, Pantoae, Proteus, Providencia, Psychrobacter and Serratia.CONCLUSIONS: High occurrence of ant (3”)c and aph (3”)c among these bacteria call for urgent attention among public health workers, because these genes can be easily disseminated to consumers of these water samples if present on mobile genetic elements like plasmids, integrons and transposons.KEYWORDS: Aminoglycoside-modifying gene, antibiotic resistance, treated water and untreated water