Objectives The research objective of this study is to enlarge and deepen the Y chromosome research on the Croatian population and enable additional insights into the population diversity and historic events that shaped the current genetic landscape of Croatia and Southeastern Europe (SEE). Materials and Methods A high‐resolution phylogenetic and phylogeographic analysis of 66 biallelic (SNPs) and 17 microsatellite (STRs) markers of the Y chromosome was performed using 720 Croatian samples. The obtained results were placed in a wider European context by comparison with ∼4450 samples from a number of other European populations. Results A high diversity of haplogroups was observed in the overall Croatian sample, and all typical European Y chromosome haplogroups with corresponding clinal patterns were observed. Three distinct genetic signals were identifiable in the Croatian paternal gene pool ‐ I2a1b‐M423, R1a1a1b1a*‐M558, and E1b1b1a1b1a‐V13 haplogroups. Discussion The analyses of the dominant and autochthonous I2a1b‐M423 lineage (>30%) suggest that SEE had a significant role in the Upper Paleolithic, the R1a1a1b1a*‐M558 lineage (19%) represents a signal from present day Slavic populations of Central Europe in the Croatian population, and the phylogeography of the E1b1b1a1b1a‐V13 clade (around 9%) implies cultural diffusion of agriculture into Europe via the Balkan Peninsula. Am. J. Hum. Biol., 2016. © 2016 Wiley Periodicals, Inc. Am. J. Hum. Biol. 28:837–845, 2016. © 2016Wiley Periodicals, Inc.