The transport of end-of-lay hens for slaughter presents a particular cause for concern in relation to hen welfare due to their less robust condition. During the period of 1 January 2010 to 31 December 2017, 17,436,074 end-of-lay hens transported for slaughter in 3,144 consignments were monitored, i.e., all hens transported from Czech farms to slaughterhouses in the Czech Republic in the monitored period. The overall mortality of hens during transport for slaughter was 0.516%. A significant (P < 0.05) impact of outside temperature and month of the year on transport-related mortality was found. Lower outside temperature was connected with the increased hen mortality and vice versa. Correspondingly, more deaths of end-of-lay hens occurred in the cold winter months of January (0.717%) and December (0.695%); on the contrary, the lowest death rates were recorded in August (0.364%). Differences were also found when comparing transport-related mortality rates according to the transport distance. The lowest mortality (0.338%) was found in hens transported for distances up to 50 km; longer distances were associated with increasing (P < 0.05) death rates, with the greatest losses (0.801%) recorded for distances from 201 to 300 km. These findings document the need for increased care for end-of-lay hens during their transport for slaughter in the winter at lower outside transport temperatures, in particular below 0°C (e.g., by adequate temperature regulation in the means of transport), and of hens transported over longer distances (if the transport distance cannot be reduced).