In this study, 145 poplar (Populus × euramericana cv.’74/76’) seedlings, a common plantation tree species in China, were selected and their ultrasonic velocities were measured at four timepoints during the first growth year. After that, 60 poplar seedlings were randomly selected and cut down to determine their acoustic velocity, using the acoustic resonance method. The effects of influencing factors such as wood green density, microfibril angle, growth days, and root-collar diameter on acoustic speed in seedlings and the relationship between ultrasonic speed and acoustic resonance speed were investigated and analyzed in this work. The number of specimens used for investigating growth days and root-collar diameter was 145 in both cases, while 60 and two specimens were used for investigating wood density and the microfibril angle, respectively. The results of this study showed that the ultrasonic speed of poplar seedlings significantly and linearly increased with growth days, within 209 growing days. The ultrasonic velocity of poplar seedlings has a high and positive correlation with growth days, and the correlation was 0.99. However, no significant relationship was found between the ultrasonic velocity and root-collar diameter of poplar seedlings. Furthermore, a low and negative relationship was found between wood density and ultrasonic speed (R2 = 0.26). However, ultrasonic velocity significantly decreased with increasing microfibril angle (MFA) in two seedlings, and thus MFA may have an impact on ultrasonic speed in poplar seedlings. In addition, ultrasonic velocity was found to have a strong correlation with acoustic resonance velocity (R2 = 0.81) and a good correlation, R2 = 0.75, was also found between the dynamic moduli of elasticity from ultrasonic and acoustic resonance tests. The results of this study indicate that the ultrasonic technique can possibly be used to measure the ultrasound speed of young seedlings, and thus early screen seedlings for their stiffness properties in the future.