The Lions – who play in the Northern Premier League Premier Division – had made a bright start with Elliot Owen and Oliver Brown both going close before midfielder Jack Emmett saw his header cleared off the line just before the break.
Sutton improved in the second half, with defender Ben Brookes firing across the face of goal and Temi Babalola heading over from a corner.
Guiseley, though, secured a memorable cup win when Thewlis slotted in from the spot in the fourth minute of added time.
The Yorkshire Post photographer Jonathan Gawthorpe captured the best of the action:
5. Guiseley AFC v Sutton United
FA Cup 1st Round. Guiseley AFC v Sutton United Guiseley's manager Ian Richards Picture Jonathan Gawthorpe 7th November 2026. Photo: Jonathan Gawthorpe
6. Guiseley AFC v Sutton United
FA Cup 1st Round. Guiseley AFC v Sutton United Guiseley's manager Ian Richards Picture Jonathan Gawthorpe 7th November 2026. Photo: Jonathan Gawthorpe
7. Guiseley AFC v Sutton United
FA Cup 1st Round. Guiseley AFC v Sutton United Guiseley's Jack Hall celebrates at fulltime. Picture Jonathan Gawthorpe 7th November 2026. Photo: Jonathan Gawthorpe
8. Guiseley AFC v Sutton United
FA Cup 1st Round. Guiseley AFC v Sutton United Guiseley's manager Ian Richards hugs match winner Jordan Thewlis at fulltime. Picture Jonathan Gawthorpe 7th November 2026. Photo: Jonathan Gawthorpe