Lambert, 31, wins his first international call-up after scoring 15 goals in 35 Premier League starts for Southampton last season.
The striker's elevation to the England squad is unusual given he has only played one season of top-flight football and prior to Southampton represented Bristol Rovers, Rochdale, Stockport, Macclesfield and Blackpool in the lower leagues.
Manchester United striker Wayne Rooney is also included in the squad for Wednesday's game at Wembley despite missing United's pre-season with hamstring and shoulder injuries.
Old Trafford colleague Wilfried Zaha, hoping to win his second cap, makes the squad after a promising summer on tour with United and having featured in England Under-21s' disappointing European Championship campaign.
Otherwise Roy Hodgson's squad has a very settled look about it for the first meeting of England and Scotland since 1999 and the play-off for Euro 2000.
England squad to face Scotland:
Goalkeepers: Ben Foster, Joe Hart, John Ruddy
Defenders: Leighton Baines, Gary Cahill, Ashley Cole, Phil Jagielka, Glen Johnson, Phil Jones, Chris Smalling, Kyle Walker
Midfielders: Michael Carrick, Tom Cleverley, Steven Gerrard, Frank Lampard, Jack Wilshere, James Milner, Alex Oxlade-Chamberlain, Theo Walcott, Ashley Young, Wilfried Zaha,
Forwards: Jermain Defoe, Rickie Lambert, Wayne Rooney, Danny Welbeck