Pre-strain is a key factor affecting the critical conditions for hydrogen-induced delayed cracking (HIDC). In this study, the threshold stress and critical hydrogen concentration for HIDC of MS1300 and MS1500 steels with different pre-strains were measured by constant load tensile (CLT) tests. The results demonstrated that although MS1500 has higher strength, its safe service strength is lower than that of MS1300 when the hydrogen content exceeds 0.5 wppm. Additionally, the hydrogen penetration, thermal desorption spectroscopy, and hydrogen microprinting methods were also used to analyze the hydrogen-induced failure mechanism. These studies provide an effective data reference for improving the industrial production process.