Abstract: Objective: To study the influence of KLF1 gene mutations on fetal hemoglobin (Hb F) levels and their effects on thalassemia. Methods: Cases undergoing thalassemia screening at the First Affiliated Hospital of Guangxi Medical University from March 2023 to December 2024 were selected. Hb analysis and blood routine analysis were performed using a Hb analyzer and a blood cell analyzer. Sanger sequencing was used to detect KLF1 gene mutations and γ -globin gene promoter region mutations, while fluorescence PCR melting curve method and Gap-PCR were used to detect the genotypes of α- and β-thalassemia gene mutations. Results: Among 100 cases with elevated Hb F, 25 cases were detected with KLF1 gene mutations. Hb analysis showed that the Hb F levels in the 25 cases ranged from 4.9% to 75.9%, and the Hb A₂ levels ranged from 1.2% to 6.3%. Blood routine results showed that the Hb levels ranged from 63.4 g/L to 144.00 g/L, the red blood cell count (RBC) ranged from 1.90×10¹²/L to 6.48×10¹²/L, the mean corpuscular volume (MCV) ranged from 44.20 fL to 102.90 fL, and the mean corpuscular hemoglobin (MCH) ranged from 13.70 pg to 35.40 pg. Gene mutation analysis identified 5 types of KLF1 gene mutations, including c. 325 C＞T heterozygote, c. 519-525dup CGGCGCC heterozygote, c.304T＞C heterozygote and homozygote, c.1022G＞A heterozygote, and c.939G＞T heterozygote. Among the 25 cases, 14 cases were compounded with β-thalassemia and 7 cases were compounded with α-thalassemia. Conclusion: It is the first time in China that rare KLF1 gene mutations c.325 C＞T and c.939G＞T have been found to cause elevated Hb F levels, and they can alleviate the severity of anemia when combined with thalassemia. The KLF1 gene mutations c.519-525dup CGGCGCC, c.304T＞C, and c.1022G＞A have the same effects.