| Summary: | <i>Pseudomonas aeruginosa</i> is a ubiquitous opportunistic bacterium that causes diseases in animals and humans. This study aimed to investigate the genetic diversity, antimicrobial resistance, biofilm formation, and virulence and antibiotic resistance genes of <i>P. aeruginosa</i> isolated from the uterus of cow, camel, and mare with clinical endometritis and their drinking water. Among the 180 uterine swabs and 90 drinking water samples analysed, 54 (20%) <i>P. aeruginosa</i> isolates were recovered. Isolates were identified biochemically to the genus level by the automated Vitek 2 system and genetically by the amplification of the <i>gyrB</i> gene and the sequencing of the <i>16S rRNA</i> gene. Multilocus sequence typing identified ten different sequence types for the <i>P. aeruginosa</i> isolates. The identification of ST2012 was significantly (<i>p</i> ≤ 0.05) higher than that of ST296, ST308, ST111, and ST241. The isolates exhibited significantly (<i>p</i> ≤ 0.05) increased resistance to piperacillin (77.8%), ciprofloxacin (59.3%), gentamicin (50%), and ceftazidime (38.9%). Eight (14.8%) isolates showed resistance to imipenem; however, none of the isolates showed resistance to colistin. Multidrug resistance (MDR) was observed in 24 isolates (44.4%) with a multiple antibiotic resistance index ranging from 0.44 to 0.77. MDR was identified in 30 (33.3%) isolates. Furthermore, 38.8% and 9.2% of the isolates exhibited a positive extended-spectrum-β-lactamase (ESBL) and metallo-β-lactamase (MBL) phenotype, respectively. The most prevalent β-lactamase encoding genes were <i>bla<sub>TEM</sub></i> and <i>bla<sub>CTX-M</sub></i>, however, the <i>bla<sub>IPM</sub></i> gene was not detected in any of the isolates. Biofilm formation was observed in 49 (90.7%) isolates classified as: 11.1% weak biofilm producers; 38.9% moderate biofilm producers; 40.7% strong biofilm producers. A positive correlation was observed between the MAR index and biofilm formation. In conclusion, the results highlighted that farm animals with clinical endometritis could act as a reservoir for MDR and virulent <i>P. aeruginosa</i>. The emergence of ESBLs and MBLs producing <i>P. aeruginosa</i> in different farm animals is a public health concern. Therefore, surveillance programs to monitor and control MDR <i>P. aeruginosa</i> in animals are required.
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