Objective: To compare the clinical characteristics, treatment timelines, and short-term outcomes of patients with acute ischemic stroke (AIS) who developed stroke at extremely high altitude or in lowland areas and received intravenous thrombolysis, and to investigate factors associated with failure to achieve the recommended door-to-needle time (DNT). Methods: This two-center retrospective cohort study included consecutive patients with AIS treated with intravenous alteplase between January 2019 and February 2026 at Xizang Autonomous Region People’s Hospital and West China Hospital, Sichuan University. Demographic and clinical data, laboratory findings, onset-to-door time, DNT, onset-to-needle time, and in-hospital outcomes were collected. Binary logistic regression was used to identify factors associated with DNT > 60 min. Results: Among 348 patients, 170 were in the high-altitude group and 178 in the lowland group. High-altitude patients were younger, had higher rates of smoking and alcohol consumption, lower oxygen saturation, and higher red blood cell counts and hemoglobin levels (all p < 0.001). DNT was significantly longer at high altitude (p < 0.001). No significant differences were found in discharge NIHSS scores, early neurological improvement, favorable functional outcome, hemorrhagic transformation, or in-hospital mortality (all p > 0.05), although the study was underpowered to detect clinically meaningful differences in hemorrhagic transformation and in-hospital mortality. High-altitude setting remained independently associated with DNT > 60 min after adjustment (aOR, 5.381; 95% CI, 3.068–9.438; p < 0.001), reflecting a composite of altitude-related and center-specific factors. Conclusions: This study provides real-world clinical evidence on intravenous alteplase thrombolysis for patients with AIS at high altitude and helps address the limited efficacy and safety data available for high-altitude populations in previous thrombolysis studies. The comparable short-term neurological outcomes suggest that thrombolysis is feasible in this setting, although the limited statistical power for secondary safety outcomes warrants confirmation in larger studies. The longer DNT observed at the high-altitude center likely reflects both altitude-related and center-specific operational factors, and further research is needed to clarify their relative contributions. These findings may provide a clinical basis for assessing the applicability of reperfusion strategies in the unique high-altitude environment.