Lyophilization of avian semen is a new method for gene pool preservation. The goal of this study was to develop a protocol for the lyophilization of rooster semen with preserved fertility. Red Rhode Island rooster ejaculates (n = 20) were assessed by volume, motility, and concentration of spermatozoa. They were pooled and diluted 1:1 with a medium LCM-T20 containing trehalose (9.5 mM), exposed at 5 °C for 40 min and centrifuged, and then the supernatant was removed. Media LCM-T with trehalose (1.75 M) was added and exposed for 10 min. The semen was frozen in a thin layer in glass vials. Samples were lyophilized for 2 h at −150 … -50 °C. The water content of the samples after lyophilization was 6.1 ± 0.5% (CV 20%). The sample was rehydrated with a medium LCM-GA5 containing hyaluronic acid (40mg/100 mL media). The total motility of the spermatozoa was 1.0 ± 0.3%. From artificial insemination of virgin hens (n = 12) with rehydrated semen, one fertilized egg was obtained from eight laid eggs. All samples of perivitelline membranes of the obtained eggs had points of interaction with the spermatozoa (7–37 pcs/cm²), which confirmed the presence of viable rehydrated spermatozoa in the genital tract of the hen. To create a dry biobank for poultry, the first protocol for lyophilization of rooster semen was developed to ensure sperm fertility in vivo.