Background: Linezolid abuse has led to the emergence of linezolid-resistant strains. cfr, optrA, and poxtA are among the most important resistance-determining genes. Objective: To detect the antibiotic susceptibility patterns of community-acquired (CA) and hospital-acquired (HA) Gram-positive isolates in Tanta University Hospitals, and screen linezolid-resistant isolates for cfr, optrA, and poxtA genes. Methodology: Subjects recruited in this study were 168, and 232 patients with CA and HA infections, respectively. The specimens were collected from various sources, including skin and subcutaneous tissue infections, respiratory tract infections, empyema, peritonitis, urinary tract infections and bacteremia. Assessment of antibiotic susceptibility profiles of the Gram-positive isolates against cefoxitin, ciprofloxacin, erythromycin, gentamicin, linezolid, oxacillin, penicillin, quinupristin/dalfopristin, tigecycline, and vancomycin was done by disc diffusion method. Then, conventional polymerase chain reaction for cfr, optrA, and poxtA was done in case of linezolid-resistant isolates. Results: The number of Gram-positive isolates were 73 from CA infections and 73 from HA infections. Two HA isolates were linezolid-resistant; both were cfr-positive, optrA- and poxtA-negative. Conclusions: Linezolid resistance was detected only in HA Gram-positive isolates at a percentage of 2.74%. Only cfr gene was detected in linezolid-resistant isolates.