The Prime Minister expressed his “deep concern” about Russia massing an estimated 100,000 troops on the Ukrainian border when they spoke on Wednesday afternoon.
Downing Street said Mr Johnson stressed during a call lasting around 45 minutes that Ukraine has a right to aspire to Nato membership, a move strongly opposed by Mr Putin.
But No 10’s version of the call did contain positives, with the pair said to have “agreed that aggravation was in no-one’s interest” as they discussed dialogue and diplomacy.
Mr Johnson had been due to speak to the Russian leader on Monday, but was forced to delay it as he dealt with the fallout from Sue Gray’s report into parties in Downing Street.
In a statement, the Prime Minister said he expressed his “deep concern about Russia’s hostile activity on the Ukrainian border” during the call.
“Any further incursion into Ukrainian territory would be a tragic miscalculation,” he added.
Downing Street said Mr Johnson stressed that all European democracies have the right to aspire to Nato membership, and that the right “fully applies” to Ukraine.
With the call being characterised as constructive and thorough, a No 10 spokeswoman added that the pair welcomed communications on issues such as the climate crisis.
“They agreed to apply this spirit of dialogue to the current tensions in order to find a peaceful resolution,” she added.
The Kremlin said the leaders had a “detailed exchange of views” on the crisis but indicated Mr Putin raised concerns about Nato.
Mr Putin has said he does not intend to order an invasion, but there are more than 100,000 Russian troops on Ukraine’s eastern border, backed by tanks, artillery, helicopters and warplanes.
The Pentagon said US President Joe Biden was ordering around 2,000 more troops to Poland and Germany this week and roughly 1,000 more who are already based in Germany will go to Romania.
On Tuesday, Mr Johnson flew to Kyiv for crisis talks with Ukrainian President Volodymyr Zelensky
The Prime Minister warned that the UK and other allies would impose sweeping sanctions on Moscow “the moment the first Russian toecap crosses further into Ukrainian territory”.