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Introduction: Recent studies such as the EXTEND-IA, AcT, and ATTEST-2 have shown tenecteplase (TNK) may have potential advantages over alteplase for AIS. This study further evaluates the effectiveness and safety of TNK at 0.25 mg/kg IV for AIS and represents real world utilization covering a diverse population reinforcing the safety and efficacy of TNK compared with alteplase. Methods: The analysis used a multicenter, retrospective cohort study across 11 primary and comprehensive hospitals from (December 2019?April 2024), comparing TNK 0.25 mg/kg (max 25 mg) with standard of care alteplase among adults with AIS presenting within 4.5 hours. The primary outcome (LVO subgroup) was substantial early reperfusion prior to thrombectomy (mTICI 2b/2c/3 or absence of retrievable thrombus). Key secondary outcomes included door-to-needle time, admission, and discharge NIHSS and 90?day mRS, and safety (ICH, angioedema). Results: Among 476 AIS patients (TNK n=270; alteplase n=206), 226 had LVO (TNK n=115; alteplase n=111). Early reperfusion occurred in 14.8% with TNK vs 4.5% with alteplase (risk difference 10.3% [95% CI, 2.7?17.8], p=0.008). Adjusted odds (OR 3.53 [95% CI, 1.20?10.40], p=0.022). In all AIS patients, median door?to?needle time was shorter with TNK (34 vs 45 minutes; difference ?11 [95% CI, ?14.4 to ?7.6], p<0.001). Good 90?day functional outcome (mRS 0?2) was more common with TNK among LVO patients (47.3% vs 29.3%, p=0.031) and among all AIS patients (61.8% vs 50.0%, difference 11.8% [95% CI, ?0.3 to 23.4], p=0.044). Symptomatic ICH and angioedema were similar; across all patients combined, ICH (symptomatic and asymptomatic) was less frequent with TNK (8.5% vs 15.0%, p=0.030). Conclusion: In routine practice, TNK 0.25 mg/kg was associated with higher pre?thrombectomy reperfusion in LVO, shorter door?to?needle times, comparable safety, and improved functional outcomes versus alteplase in key analyses. These findings align with recent randomized evidence supporting TNK as an effective alternative to alteplase for AIS.