The winner of the tie will welcome either Manchester United or Derby County to their ground in the fifth round.
The U's are in fine form heading into the Colchester fixture having won their last five games, most recently beating Cheltenham Town in a 6-3 thriller last weekend.
Unbeaten in seven, the Yellows dispatched of Wigan Athletic in the third round of the cup, winning 2-0 at the Kassam with Jack Lee and Mo Missanga scoring the goals to see them through.
Forward threat Lee signed his first professional contract last summer and has impressed in front of goal this season, making sure he is someone who Oxford see as having a high ceiling with his development.
🗣️ Pre-match thoughts from Pablo Haysham and Niall Keown ahead of the #FAYouthCup! pic.twitter.com/4K1ltdo7LS
— Oxford United Academy (@AcademyOUFC) January 21, 2026
The U's next opponents, Colchester, beat Halifax Town in the previous round with a 1-0 victory away from home.
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Ahead of the big cup game this evening, U18s head coach Niall Keown told the club website: "Overall, looking back at the (Wigan) game I was really pleased with how the boys did on the night and how they dealt with the occasion.
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"I think that was one of the main things going into the game is with it being pretty much for all of them the first time playing at the Kassam and for a lot of them the first time playing in a Youth Cup.
"We know that there's still a long way for them to go in terms of their development and they've got to keep pushing in the way that they have at the start of this season to give themselves the best possible chance of succeeding longer term."
Tickets remain on sale for the FA Youth Cup fixture at the Kassam, with a victory setting up the potential visit of giants Manchester United in the next round.