U21s host Shrimpers on Tuesday.
Summer signings Ben Wyatt, Dexter Peter and Courtney Senior all play against the Shrimpers in the match at the U's training ground, which kicks off at 1pm.
The two senior teams went head to head at the weekend and Tuesday will see the younger players given a chance to impress boss Wayne Brown ahead of the new season.
Colchester United's U21s will face their Southend counterparts on Tuesday afternoon at Florence Park.
U's: Barnes, Bransgrove, O'Donoghue, Wilson, Wyatt, Harney, Peter, Elokobi, James, Diaz Wright, Edge, Issa, Senior, Brown, Curtis, Sheriff, Regis