Kane picked up the problem in the process of scoring an offside goal and left St Mary's Stadium on crutches, sparking fears he could be set for a lengthy spell on the sidelines.
Head coach Jose Mourinho said it was too soon to know how bad the injury is but described it as "negative".
"For Harry to come out is negative," he said. "Hamstring is always a negative. Is it tear? Is it a spasm? Is it a contraction? In this moment I cannot say."
Spurs are now set to investigate the severity of the problem and he will now most likely undergo a scan to determine how serious the problem is.
Kane was not the only player Spurs lost to injury as record signing Tanguy Ndombele limped off in the first half and it caused Mourinho to lose his patience.
The France international has been beset by fitness concerns following his summer move from Lyon and recently told his manager he did not want to be selected for the Boxing Day win over Brighton.
Mourinho did not hold back in his assessment of the Frenchman.
"He is always injured, he is injured, is not injured," Mourinho said. "He plays one match, the next week he is injured, he plays another match. We are full of hopes and this is since the beginning of the season."
The injuries will only worsened the mood of Mourinho, who received a yellow card from referee Mike Dean following an altercation with Saints goalkeeping coach Andrew Sparkes.
Mourinho is understood to have taken exception to some time-wasting antics and later branded Sparkes "an idiot".
Asked about the incident after the game, Mourinho said: "I think the yellow card is fair because I was rude, but I was rude to an idiot."