The visitors reduced the gap to 27-21 early in the second half, but a yellow card to pivotal first-five Fletcher Smith for an offside infringement saw Otago extend their lead, and two late Waikato tries only made the margin more respectable.
The defeat also sees Waikato miss out on a chance to move into the top four in the Premiership, instead still sitting three points behind Auckland with two games to go.
Otago remain third in the Championship and look set for a semifinal berth, though by beating Canterbury they could lock in that accomplishment - and lock away the Shield for the summer.
Otago 45 (Vilimoni Koroi 2, Slade McDowall, Jona Nareki, Henry Purdy, Aleki Morris-Lome tries; Josh Ioane 3 cons, 3 pens)
Waikato 35 (Samisoni Taukei'aho 2, penalty try, Mitchell Jacobson, Simon Parker tries; Fletcher Smith 4 cons)
Otago v Waikato as it happened: