The influence of microstructure homogeneity on crack intiation behavior under high-cycle fatigue (HCF) test at room temperature was studied in Ti-55531 alloy with lamellar microstructure by TEM and SEM methods. The results show that the HCF strength of Ti-55531 alloy could be as high as 639 MPa. However, a small amount of grain boundary (GB) α and a large number of inhomogenious regions in microstructure significantly effect the HCF behavior of lamellar Ti-55531 alloy. Fatigue microcracks mainly initiated at interfaces between αs and β phases owing to their different structure and properties. Moreover, small amount of microcracks initiated at GB α and αs phases during fatigue.