Since the introduction of artificial insemination, Holstein bulls have been reduced to 2 Y-chromosome lineages. One possible reason is that modern Y-lineages (MY) had better semen quality than lost Y-chromosome lineages (LY). The objective of this study is to compare semen quality of young bulls from LY to semen quality in MY. Two LY were resurrected through in vitro embryo production using semen from ZIMMERMAN ALSTAR PILOT (born in 1954) and U-OF-MINN W CALIBN CUTHBERT, who was a ROSAFE CALIBAN (born in 1953) son born during a long-term selection experiment; semen from both bulls was procured from the National Animal Germplasm Program repository. Two sons of CUTHBERT born in March 2017 and 3 sons of PILOT born in November 2017 were transferred to Select Sires Inc. Semen was collected in the summer of 2018 and in January 2019, respectively. Two ejaculates were taken each date of collection and measured for semen volume, sperm concentration, total number of sperm, and visually scored motility. The 5 LY bulls were compared with 58 age-matched MY bulls collected at the same time. Analyses were conducted to compare fixed effects of LY with MY, or with each Y-chromosome lineage treated as a separate effect (n = 4) in a mixed model. The explanatory variables considered were fixed effects of week of year, daily ejaculate number (1 or 2), and interval between collection days; bull identity was included as a random effect. Interval between collections was not significant for any trait and ejaculate number was not significant for motility. Significant (P < 0.05) differences between LY and MY were evident for total number of sperm (4.6 billion versus 3.4 billion, respectively) and sperm concentration (1.3 billion/ml versus 1.0 billion/ml, respectively). For motility, Y-lineage treated as a separate effect tended toward significance (P < 0.10) with CUTHBERT sons (74.8%) having lower motility than sons of the MY bull ELEVATION (77.3%) and PILOT (77.6%). There were no significant differences among lineages for semen volume. This research suggests that initial semen quality may not be the primary reason Y-chromosome lineages were lost from the Holstein breed.