The England captain equalled Peter Shilton’s record for the most appearances by an England men’s international and produced another outstanding display to strengthen Thomas Tuchel’s side’s position in Group A3.
Coufal own goal puts England ahead
England dominated possession from the opening stages but had to wait until the 27th minute to break the deadlock.
Kane played a dangerous cross into the Czechia penalty area, and Vladimír Coufal turned the ball into his own net under pressure to give England a 1-0 lead.
The goal rewarded England’s sustained pressure and allowed Tuchel’s side to play with even greater confidence.
Kane doubles England’s advantage
Kane then got his own name on the scoresheet shortly before half-time.
Elliot Anderson produced an excellent ball forward before Morgan Rogers delivered a first-time cross into the penalty area.
Kane found space in front of goal and finished clinically to send England into the interval 2-0 ahead.
It was a fitting way for the captain to mark his landmark 125th appearance.
Kane completes his brace
England continued to control proceedings after the restart and Kane completed his double in the 57th minute.
Lewis Hall delivered a corner into the penalty area, where Kane rose above the Czech defence and powered a header beyond goalkeeper Lukáš Horníček.
The goal took Kane's international tally to an extraordinary 91 goals for England.
England 3-0 Czechia
• England: Vladimír Coufal own goal 27', Harry Kane 40', 57'
• Czechia: No goals
Kane equals Shilton's record
The biggest individual storyline of the night was Kane's historic appearance.
The 33-year-old Bayern Munich striker won his 125th England cap, drawing level with Shilton, whose record had stood since 1990.
Kane has already surpassed Wayne Rooney as England's leading goalscorer and now stands alone alongside Shilton at the top of the appearance list. He will have the opportunity to become England's outright record appearance holder during the November international window.
England strengthen their position in Group A3
The victory moved England to nine points from four matches, leaving them second behind Spain, who have 12 points after beating Croatia 2-1.
Czechia remain bottom of the group without a point, while Croatia have three points.
England have now responded strongly after their opening 3-2 defeat to Spain, following it with three consecutive victories, including a 2-0 win over Czechia in Prague and Saturday's emphatic 7-0 demolition of Croatia in Rijeka.
Tuchel's side close international break in style
The victory completes an impressive four-match Nations League window for England.
Although Spain remain top of Group A3, England are now all but assured of a place in the quarter-finals, with the top two teams progressing to the knockout stage.
For Kane, meanwhile, it was another landmark evening in an extraordinary international career — one capped by a record-equalling appearance and a decisive brace at Wembley.