Sutton United and Boreham Wood will join six other non-Scottish sides in the second round of next season's Scottish Challenge Cup.
Northern Irish and Welsh teams have featured in the past two seasons and two from Republic of Ireland entered in season 2017-18.
And now the National League teams will take part.
Scottish Premiership under-20s sides, or 'colt teams', also compete with Championship, League One and Two sides.
Inverness Caledonian Thistle won the most recent staging of the tournament, beating Dumbarton in the March final.
The draw for the first round, which will feature just Scottish sides including the colt teams, will take place on 26 June.
There will also be a preliminary round in which the third and fourth-placed sides in the Scottish Highland and Lowland Leagues will compete to join the winners and runners-up from those leagues in the first round.
Crusaders and Coleraine will be the Northern Irish sides in round two, with Bohemians and Bray Wanderers representing Republic of Ireland and the New Saints and Connah's Quay Nomads the Welsh entrants.
The Scottish Professional Football League said Sutton and Boreham were selected as "the highest-ranked Vanarama National League sides in 2017-18 still competing in the division".
"The Irn-Bru Cup remains an innovative and evolving competition, and we are pleased to now extend a warm welcome to the National League and their representatives, Sutton United and Boreham Wood," said SPFL chief executive Neil Doncaster.
"We look forward to developing a positive relationship with our counterparts in England, just as we have with representatives from the leagues of Northern Ireland, Wales and the Republic of Ireland over the last two years."
Preliminary round (two ties) - 31 July & 1 August
First round (24 ties) - 14/15 August
Second round (16 ties) - 8/9 September
Third round (eight ties) - 13/14 October
Quarter-finals - 17/18 November
Semi-finals - 16/17 February
Final - 24 March